CHARACTERISATION OF EPIDERMAL TIGHT JUNCTION PROTEINS IN AGEING
A thesis submitted to The University of Manchester for the degree of PhD in the Faculty of Medical and Human Sciences
2012
SUHA M ALTHUBAITI
School of Medicine
LIST OF CONTENTS
LIST OF TABLES ...... 6
LIST OF FIGURES ...... 7
ABSTRACT ...... 10
DECLARATION ...... 11
COPYRIGHT STATEMENT ...... 12
ACKNOWLEDGEMETS ...... 13
PUBLICATIONS ...... 14
LIST OF ABBREVIATIONS ...... 15
1. INTRODUCTION...... 17
1.1 Structure and functions of skin ...... 17
1.1.1 Basic structure of skin ...... 18 1.1.2 Epidermal structure ...... 20 1.2 Tight junctions – structure and function ...... 22
1.2.1 Other functions of tight junctions...... 25 1.2.2 Molecular composition of tight junctions ...... 26 1.2.3 Transmembrane components...... 26 1.2.4 Other transmembrane proteins ...... 30 1.2.5 Cytosolic proteins ...... 31 1.3 The role of tight junctions in the skin ...... 32
1.3.1 Non-Junctional expression of tight junction proteins ...... 35 1.4 Ageing and skin ...... 35
1.4.1 Intrinsic ageing ...... 39 1.4.2 Photoageing ...... 40 1.4.3 Acute effects of ultraviolet radiation on skin ...... 43 1.5 Epidermal barrier function and Ultraviolet radiation ...... 44
1.6 The role of tight junctions in age related changes ...... 46
Hypothesis, aims and objectives: ...... 47 2
2. MATERIAL AND METHODS ...... 49
2.1 Materials ...... 49
2.2 Patient Samples ...... 49
2.2.1 Intrinsic ageing study ...... 50 2.2.2 Photoageing study ...... 50 2.2.3 Acute UV irradiation of human subjects ...... 50 2.3 Histology ...... 52
2.3.1 Sectioning of tissue ...... 52 2.4 Immunostaining of human skin ...... 52
2.5 Cell culture ...... 55
2.5.1 Recovering cells from liquid nitrogen ...... 56 2.6 Ultraviolet irradiation of cells ...... 56
2.7 Measurement of cell viability...... 58
2.7.1 MTT assay ...... 58 2.8 Measurement of Trans-epithelial Electrical Resistance (TEER) ...... 58
2.9 Preparation of protein extracts ...... 58
2.10 Determination of protein concentration ...... 59
2.11 SDS-PAGE (Sodium dodecyl sulfate polyacrylamide gel electrophoresis) and Western blotting ...... 61
2.12 Immunofluorescent staining of keratinocytes ...... 61
2.13 Quantitative Reverse Transcription Polymerase Chain Reaction (qRT- PCR)...... 62
2.14 Data presentation and statistical analysis...... 63
3. A COMPARISON OF TIGHT JUNCTION PROTEIN EXPRESSION IN INTRINSICALLY AGED VS YOUNG HUMAN SKIN ...... 64
3.1 Introduction ...... 64
3.2 Results ...... 65
3.2.1 Analysis of Affymetrix® data ...... 65
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3.2.2 Detection and quantitation of tight junction proteins using immunofluorescent staining…...... 67 3.2.3 Comparison of tight junction protein expression in old vs. young human skin……...... 79 3.2.4 A slight reduction of epidermal thickness in aged vs young human skin ..... 87 3.3 Discussion ...... 88
4. AN INVISTIGATION INTO THE EXPRESSION OF TIGHT JUNCTION PROTEINS IN PHOTOAGED VS. PHOTOPROTECTED HUMAN SKIN ...... 93
4.1 Introduction ...... 93
4.2 Results ...... 94
4.2.1 Claudin-1 expression is significantly decreased in photoaged skin ...... 94 4.2.2 Claudin-7 is significantly increased in photoaged skin ...... 97 4.2.3 Claudin-12 is significantly increased in photoaged skin ...... 98 4.2.4 No significant changes are observed in other tight junction proteins in photoaged skin………...... 100 4.2.5 A reduction in claudin-1 and an increase in claudin-12 mRNA levels is observed in photoaged human skin ...... 100 4.3 Discussion ...... 102
4.3.1 Expression and localisation of claudins-1, -7 and -12 in photoprotected and photoaged skin ...... 102 5. CHARACTERISATION OF TIGHT JUNCTION PROTEIN EXPRESSION IN HUMAN SKIN SUBJECTED TO A SINGLE, ACUTE DOSE OF UVB ...... 106
5.1 Introduction ...... 106
5.2 Results ...... 107
5.2.1 The expression of claudin-1 is significantly reduced in response to a single dose of UVB………...... 107 5.2.2 The expression of claudins-7 and -12 are unaltered in response to a single dose of UVB………...... 109 5.3 Discussion ...... 112
6. ULTRAVIOLET-B IRRADIATION MODULATES TIGHT JUNCTION PROTEIN EXPRESSION IN CULTURED PRIMARY KERATINOCYTES ...... 116
6.1 Introduction ...... 116
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6.2 Results ...... 117
6.2.1 UVB irradiation of undifferentiated NHEK alters the expression of tight junction proteins…...... 117 6.2.2 Tight junction protein expression does not recover completely over time . 120 6.2.3 UVR does not affect the expression of tight junction protein mRNA ...... 120 6.2.4 Differentiated keratinocytes showed no significant changes in tight junction protein expression in response to UVR ...... 122 6.2.5 UVR causes reductions in the barrier function of keratinocytes ...... 126 6.2.6 UVR induces disruptions to the localisation of tight junction proteins in keratinocytes ...... 128 6.3 Discussion ...... 133
7. GENERAL CONCLUSIONS AND FURTHER WORK ...... 137
Strengths and Weaknesses -Further Work ...... 142
APPENDICES ...... 144
REFERENCES ...... 147
Word Count: 30,708 words.
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LIST OF TABLES
Table 1.1 Components and systems of the skin: functions and changes with age...... 36
Table 2.1 Fixation methods of tight junction proteins and dilutions of primary and secondary antibodies used in immunostaining...... 54
Table 2.2 Standard curve standards. A standard curve for the BCA protein assay composed of 7 standards...... 60
Table 3.1 Affymetrix® genome array of TJ...... 66
Table 5.1 The MED values for acutely irradiated human skin...... 112
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LIST OF FIGURES
Figure 1.1 The structure of skin...... 19
Figure 1.2 The structure of the epidermis...... 21
Figure 1.3 Schematic diagram of the junctional complex and tight junctions...... 23
Figure 1.4 Schematic representation of the tight junction's basic structural components and the paracellular and transcellular pathways of transport across the epithelium...... 25
Figure 1.5 The interactions between loops of clds in the intracellular space providing a paracellular seal...... 29
Figure 1.6 The clinical differences between sun exposed area (photoageing) and sun protected area (chronological ageing)...... 38
Figure 1.7 Hypothetical model of the dermal damage and photoageing caused by UV radiation...... 42
Figure 2.1 Diagram of immunofluorescent staining intensity measurement using ImageJ software...... 55
Figure 2.2 Relative spectral output of TL-12 source...... 57
Figure 2.3 BCA Standard curve...... 60
Figure 3.1a The optimisation method of the immunostaining of cld-1 TJ protein in human buttock epidermis...... 69
Figure 3.1b The optimisation method of the immunostaining of cld-4 TJ protein in human buttock epidermis...... 70
Figure 3.1c The optimisation method of the immunostaining of cld-5 TJ protein in human buttock epidermis...... 71
Figure 3.1d The optimisation method of the immunostaining of cld-7 TJ protein in human buttock epidermis...... 72
Figure 3.1e The optimisation method of the immunostaining of cld-8 TJ protein in human buttock epidermis...... 73
Figure 3.1f The optimisation method of the immunostaining of cld-12 TJ protein in human buttock epidermis...... 74
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Figure 3.1g The optimisation method of the immunostaining of occludin TJ protein in human buttock epidermis...... 75
Figure 3.1h The optimisation method of the immunostaining of zo-1 TJ protein in human buttock epidermis...... 76
Figure 3.2 Localisation of TJ proteins in human skin...... 78
Figure 3.3a Immunofluorescent staining of cld-1 TJ protein shows no significant changes between young vs. intrinsically aged human skin...... 80
Figure 3.3b Immunofluorescent staining of cld-4 TJ protein shows no significant changes between young vs. intrinsically aged human skin...... 81
Figure 3.3c Immunofluorescent staining of cld-5 TJ protein shows no significant changes between young vs. intrinsically aged human skin...... 82
Figure 3.3d Immunofluorescent staining of cld-7 TJ protein shows no significant changes between young vs. intrinsically aged human skin...... 83
Figure 3.3e Immunofluorescent staining of cld-8 TJ protein shows no significant changes between young vs. intrinsically aged human skin...... 84
Figure 3.3f Immunofluorescent staining of cld-12 TJ protein shows no significant changes between young vs. intrinsically aged human skin...... 85
Figure 3.3g Immunofluorescent staining of occludin TJ protein shows no significant changes between young vs. intrinsically aged human skin...... 86
Figure 3.3h Immunofluorescent staining of zo-1 TJ protein shows no significant changes between young vs. intrinsically aged human skin...... 87
Figure 3.4 Epidermal thickness comparison between young and aged human epidermis...88
Figure 4.1 Claudin-1 expression is decreased in photoexposed vs photoprotected skin.. .. 96
Figure 4.2 A reduction in the expression of cld-1 in the basal layer in photoaged skin.. ... 97
Figure 4.3 Claudin-7 is increased in photoaged skin...... 98
Figure 4.4 Intensity of cld-12 staining is significantly increased in photoaged skin...... 99
Figure 4.5 Quantitative PCR analysis of TJ proteins clds-1 and -12 showed changes in their expression level in photoaged human skin...... 101
Figure 5.1 Claudin-1 expression is decreased following acute UVB irradiation of human skin...... 108
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Figure 5.2 Claudin-1 expression is significantly decreased in the basal layer following acute UVB irradiation...... 109
Figure 5.3 UVB irradiation does not significantly alter cld-7 expression...... 110
Figure 5.4 UVB irradiation does not significantly alter cld-12 expression...... 111
Figure 6.1 UVR at doses below 20mJ/cm2 does not affect the viability of primary keratinocytes...... 118
Figure 6.2 Tight junction protein expression in response to UVR in undifferentiated keratinocytes...... 119
Figure 6.3 The expression of clds-1 and -4 does not return to baseline levels within 96 hours of irradiation...... 121
Figure 6.4 Quantitative PCR analysis of TJ proteins clds-1 and -4 showed no changes in their expression in irradiated keratinocytes...... 122
Figure 6.5 UVR at doses below 20mJ/cm2 does not significantly affect the viability of differentiated keratinocytes...... 123
Figure 6.6 UVR does not affect expression of TJ proteins in differentiated keratinocytes...... 124
Figure 6.7 Calcium switch abolishes the effects of UVR on TJ protein expression in keratinocytes...... 125
Figure 6.8 UVR disrupts TJ function depending on the timing of irradiation...... 127
Figure 6.9a The effect of UV irradiation on the organization of cld-1 TJ protein in NHEK cells...... 129
Figure 6.9b The effect of UV irradiation on the organisation of cld-4 TJ protein in NHEK cells……………………………………………………………………………………….130
Figure 6.9c The effect of UV irradiation on the organisation of Occludin TJ protein in NHEK cells...... 131
Figure 6.9d The effect of UV irradiation on the organisation of Zo-1 TJ protein in NHEK cells...... 132
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The University of Manchester Suha M Althubaiti A thesis submitted to The University of Manchester for the degree of PhD in the Faculty of Medical and Human Sciences CHARACTERISATION OF EPIDERMAL TIGHT JUNCTION PROTEINS IN AGEING 2012
ABSTRACT
The epidermal tight junction (TJ) plays an important role as a barrier which protects the skin against dehydration and infection. Skin barrier function is known to decline with increasing age (Rabe et al., 2006). Human skin is subject to intrinsic (i.e. chronological) ageing and extrinsic (environmentally-induced) ageing. However, the role of TJs in the ageing process is still unknown. Therefore, in this study, using quantitative immunofluorescent staining TJ protein expression was investigated in intrinsically aged compared to young human skin. Since ultraviolet radiation (UVR) from sunlight is considered the major environmental insult to human skin, TJ proteins were also investigated in photoaged compared to photoprotected human skin, and in skin exposed to a single acute dose of UVR. In aged vs young skin, there was no significant difference either in the expression levels or localisation of TJ proteins. However, significant reduction in claudin-1 (cld-1) and increases in cld-7 and -12 expressions were demonstrated in chronically photoaged human skin suggesting differential regulation of clds in response to photoexposure. By contrast in acutely irradiated human skin, only a reduction in cld-1 expression was observed 24h after a single UVB dose. Moreover, in both chronic and acute UVR exposed human skin, cld-1 was most significantly reduced in the basal layer of the epidermis suggesting that the differentiation state of keratinocytes might be important in their response to UVR. To investigate these effects further, a normal human epidermal keratinocyte (NHEK) cell culture model was employed. A reduction in cld-1 expression and an increase in cld-4 were demonstrated in undifferentiated NHEK cells irradiated with sub lethal doses of UVR. Interestingly, no changes were observed in TJ protein expression in irradiated differentiated keratinocytes. However, when TJ function was measured in these cells using transepithelial electrical resistance (TEER) as a marker of TJ function, UVR induced a significant reduction in TEER. This coincided with an alteration in the organisation of cld- 1 in irradiated differentiated keratinocytes. These data demonstrate that TJ protein expression is modulated by acute and chronic exposure to UVR. These observations may explain, at least in part, the decline in skin barrier function observed in response to UVR.
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DECLARATION
No portion of the work referred to in the thesis has been submitted in support of an application for another degree or qualification of this or any other university or other institute of learning.
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COPYRIGHT STATEMENT
i. The author of this thesis (including any appendices and/or schedules to this thesis) owns certain copyright or related rights in it (the “Copyright”) and s/he has given The University of Manchester certain rights to use such Copyright, including for administrative purposes. ii. Copies of this thesis, either in full or in extracts and whether in hard or electronic copy, may be made only in accordance with the Copyright, Designs and Patents Act 1988 (as amended) and regulations issued under it or, where appropriate, in accordance with licensing agreements which the University has from time to time. This page must form part of any such copies made. iii. The ownership of certain Copyright, patents, designs, trade marks and other intellectualproperty(the“IntellectualProperty”)andanyreproductionsofcopyrightworks inthethesis,forexamplegraphsandtables(“Reproductions”),whichmaybedescribedin this thesis, may not be owned by the author and may be owned by third parties. Such Intellectual Property and Reproductions cannot and must not be made available for use without the prior written permission of the owner(s) of the relevant Intellectual Property and/or Reproductions. iv. Further information on the conditions under which disclosure, publication and commercialisation of this thesis, the Copyright and any Intellectual Property and/or Reproductions described in it may take place is available in the University IP Policy (see http://documents.manchester.ac.uk/DocuInfo.aspx?DocID=487), in any relevant Thesis restriction declarations deposited in the University Library, The University Library’s regulations (see http://www.manchester.ac.uk/library/aboutus/regulations) and in The University’spolicyonPresentationofTheses
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ACKNOWLEDGEMETS
I would like to express my deepest gratitude to mysupervisor,Dr.CatherinO’Neill for her patient guidance and constructive suggestions throughout the development of this research. I would also like to extend my gratitude to Dr. Rachel Watson, Professor
Christopher Griffiths and Dr. Neil Gibbs for their appreciated help and supervision.
I would like to acknowledge the support that King Saud bin Abdulaziz University provided me with.
Most importantly, none of this would have been possible without the support of my beloved parents, my father General Mohammed Althubaiti who supported and encouraged me all my life and to the best lady on Earth my mother Dr. Fathia for being a constant source of love and strength over the years. Also Special thanks to my wonderful sisters
Sara, Dr. Alaa, Noura and to my brothers Dr. Abdulrahman and Dr. Majed.
A very deep and warm appreciation to all my friends and close ones which I was truly blessed with and so fortunate to have them in my life.
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PUBLICATIONS
Althubaiti S., Griffiths C.E.M., Gibbs N.K., O'Neill C.A. and Watson R.E.B. (2010). The tight junction protein claudin 1 is reduced in photoaged human epidermis. British Journal of dermatology, 162 (4). eScholarID:94808
Althubaiti S., Griffiths C.E.M, Gibbs N.K., Watson R.E.B. and O'Neill C.A. (2010). The tight junction protein claudin 7 is increased in photoaged human epidermis. Journal of Investigative dermatology, 130 (2). eScholarID:94812
Althubaiti S., Griffiths C.E.M., Gibbs N.K., Watson R.E.B. and O’Neill C.A. (2011). Ultraviolet-B irradiation modulates tight junction protein expression in cultured primary keratinocytes. British journal of dermatology, 164; page 926. eScholarID:133677
Althubaiti S., Griffiths C., Gibbs N., O'Neill C. and Watson R. (2011). Characterisation of tight junction proteins in photoaged epidermis. Journal of investigative dermatology, 131(2), S106. eScholarID:133689
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LIST OF ABBREVIATIONS
AD Atopic Dermatitis AJ Adherens Junction AP-1 Activator Protein 1 AQP3 Aquaporin-3 BCA Bicinchoninic Acid BSA Bovine Serum Albumin CE Cornified Cell Envelopes clds Claudins DAPI 4',6-Diamidino-2-phenylindole DEJ Dermal-Epidermal Junction DMSO Dimethyl Sulphoxide DS Desmosome. ECM Extracellular Matrix FB Fibroblasts FITC Fluorescein Isothiocyanate GAPDH Glyceraldehyde-3- Phosphate Dehydrogenase HaCaT Human Keratinocyte Cell Line HRP Horseradish Peroxidase HS Heparan Sulfate HSE Human Skin Equivalents JAM Junctional Adhesion Molecule KBM Keratinocyte Basal Medium KC Keratinocytes MAPK Mitogen-Activated Kinase Pathway MDCK cells Madin-Darby Canine Kidney Cells MED Minimal Erythemal Dose MMP Matrix Metalloproteinase Mv Microvilli NHEK Normal Human Epidermal Keratinocytes OCT Optical Cutting Temperature 15
PBS Phosphate-Buffered Saline PFA Paraformaldehyde PKC Protein Kinase C qRT-PCR or qPCR Quantitative Reverse Transcription Polymerase Chain Reaction ROS Reactive Oxygen Species S. aureus Staphyococcus Aureus SB Stratum Basale SC Stratum Corneum SDS-PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis SG Stratum Granulosum SL Stratum Lucidum SS Stratum Spinosum TBS Tris-Buffered Saline Solution TEER Trans-Epithelial Electrical Resistance TEWL Trans-Epidermal Water Loss TIMPs Tissue Inhibitor of Matrix Metalloproteinases TJs Tight Junctions UVA Ultraviolet Radiation A UVB Ultraviolet Radiation B UVR Ultraviolet Radiation ZO Zonula Occludin
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1. INTRODUCTION
1.1 Structure and functions of skin
Skin,whichissometimesreferredtoasthe‘integumentarysystem’,isthelargest and the most obvious organ of the body. Skin accounts for approximately 15% of the total adult body weight as it covers the whole external surface of the body and the mucous membranes lining the body's orifices (Kanitakis, 2002).
The primary role of skin is to provide a barrier between the body and the outside world. This barrier prevents water and electrolyte loss from within the body and also prevents entry of toxins and bacteria from the environment. The importance of the skins barrier is demonstrated in conditions where the barrier is breached e.g. in premature babies where the barrier is incomplete (Kalia et al., 1998) or in victims of severe burns (Proksch et al., 2008). These individuals are at significant risk of dehydration and infection due to the breach in the barrier. However, since it exists at the interface of the body and the environment, skin is often subjected to environmental insults that perturb the barrier e.g. wounding (Koster, 2010). The skin has evolved a robust immune response and a remarkable ability to repair the barrier quickly so that the body is protected (Proksch et al.,
2008).
As well as its barrier function, skin also has several other important functions including regulation of temperature, sensation, excretion and synthesis of vitamin D. Skin is also important in a social context as it is part of the mechanism by which we recognise people (Tortora and Grabowski, 2001; Brown and Burns, 1996; Jowett and Ryan, 1985).
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The mechanism by which the skin fulfils all these vital functions is due to its unique structure and this is discussed below.
1.1.1 Basic structure of skin
Skin is not simply a flat organ but is made up of three layers: the epidermis, the dermis and the hypodermis (or subcutaneous layer). Each layer differs in thickness, function and mechanical strength and has its own unique structure (fig 1.1; Tortora and
Grabowski, 2001). The skin also contains appendages that traverse multiple skin layers.
Major structures include sweat glands, hair follicles, sebaceous glands and nails (Brown and Burns, 1996).
The hypodermis is the deepest layer of skin and separates the skin from muscles.
Mainly composed of adipocytes, the hypodermis is important for helping to preserve body heat. The hypodermis also contains blood vessels that help to supply nutrients to the skin
(Brown and Burns, 1996).
The dermis contains fibroblasts which produce collagen (which provides strength) and elastin (which provides elasticity). Also the dermis contains macrophages, adipocytes, neurones, sweat glands and hair roots (reviewed Naylor et al., 2011)
The epidermis is the outermost layer of skin and provides the main permeability barrier without which life as terrestrial mammals would be impossible. This permeability barrier is constantly renewed by terminally differentiating keratinocytes which comprise
90% of the cells in the epidermis. However, the epidermis also contains other cells types such as Langerhans’ cells which are vital for the immune surveillance and the pigment producing melanocytes.
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Figure 1.1 The structure of skin. Skin is composed of three layers: the epidermis, the dermis and the hypodermis (subcutaneous layer). Accessory structures of the skin include hair, nails and glands along with associated muscles and nerves (taken from Tortora and Grabowski, 2001).
The process of terminal differentiation leads to the production of a multilayered, stratified epithelium consisting of 4 main layers: stratum basale (SB), stratum spinosum
(SS), stratum granulosum (SG), and stratum corneum (SC) (fig 1.2). A fifth layer, the stratum lucidum (SL), is found at some body sites such as the soles of the feet, where wear and tear necessitates a thicker epidermis (Moran and Rowley, 1988; Brown and Burns,
1996).
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1.1.2 Epidermal structure
The stratum basale is the deepest layer of the epidermis and consists of large columnar cells which are attached to a basement membrane, the dermal-epidermal junction. These cells proliferate and provide daughter cells that undergo terminal differentiation to produce the other layers of the epidermis. Terminal differentiation begins when, through a process that is incompletely understood, basal keratinocytes migrate up to the spinous layer and in doing so begin to synthesis a completely new set of structural proteins. Hence in the spinous layer, keratins-5 and -14, the main products in proliferating basal cells, are replaced by keratins 1 and 10 which are assembled into filaments. In the spinous layer, the cells become interlocked by desmosomes which gives them a spiky appearance under the microscope. Hence, this layer is also sometimes referred to as the spinylayeror "prickle celllayer” Intheupperspinous layer, keratinocytes also contain lamellar bodies that are rich in ceramides, cholesterol and free fatty acids. These lamellar bodies persist until their exocytotic release in the upper granular layer (Moran and Rowley,
1988; Brown and Burns, 1996).
The stratum granulosum or granular layer is so-called because the keratinocytes in this layer can be seen to contain keratohyalin granules. These granules contain precursors ofthe‘cornifiedenvelope’suchasprofilaggrinandloricrin.Finallythestratum corneum is the outermost layer of the epidermis where keratinocytes begin to collapse resulting in flattened, terminally differentiated cells (Moran and Rowley, 1988; Brown and Burns,
1996; Schaefer and Redelmeier, 1996). The lipid contents of lamellar bodies are found in the intercellular spaces of the stratum corneum together with the protein contents of keratohyalin granules that are covalently cross linked by transglutaminase-1 to form cornified cell envelopes (CE) (Cartlidge, 2000; Wertzp, 2000; Elias et al., 1977; Steinert,
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2000; Schaefer and Redelmeier, 1996). This arrangement of terminally differentiated cells embedded in a lipid matrix helps to provide the skin with its permeability barrier and the barrier is constantly renewed throughout life. In healthy skin the process of terminal differentiation is highly regulated and desquamation (cell shedding at the surface) is precisely balanced by proliferation in the basal layer (Liu et al., 2009).
Figure 1.2 The structure of the epidermis. The epidermis consists of five layers: stratum basale includes Merkel cell and melanocytes; the stratum spinosum including Langerhans cells; the stratum granulosum, the stratum lucidum and the stratum corneum. (a) Shows a cartoon image of the four principle cell types in the epidermis. (b) A photomicrograph of the skin stained with haematoxylin and eosin (taken from Tortora and Grabowski, 2001).
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The stratum corneum is a major permeability barrier and indeed, diseases or gene defects that prevent its proper formation result in severe phenotypes (Ramos-E-Silva.,
2012). Therefore it is perhaps unsurprising that until recently, the stratum corneum was considered as the major permeability barrier. However, in 2002, Furuse et al produced a transgenic mouse that was deficient in claudin-1, a principle component of structures known as tight junctions (TJs). This mouse died from excessive transepidermal water loss within 24 hours of birth and provided the first evidence of the critical role of TJs in epidermal barrier function.
1.2 Tight junctions – structure and function
Much of what is currently known about TJs has come from work in simple epithelia such as the gut. In simple epithelia, adjacent cells are linked together by the junctional complex which consists of desmosomes and the adherens complex, as well as
TJs (fig 1.3a; Farquhar and Palade, 1963). Adherens junctions and desmosomes are responsible for cell adhesion whilst TJs are responsible for intercellular sealing. Tight junctions exist at the most apical end of the junctional complex where they form a physical barrier to free passage of molecules through the so called paracellular pathway i.e. the pathway between epithelial cells. This barrier maintains normal organ function and also prevents systemic contamination by microbes and toxins existing in the surrounding environment (Tsukita et al., 2001).
When viewed by electron microscopy (fig 1.3b; Tsukita et al., 2001) in the area of the TJ, the membranes of adjacent cells appear almost to fuse and the paracellular space is almost obliterated.
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Figure 1.3 Schematic diagram of the junctional complex and tight junctions. (a) Intestinal epithelial cells are represented in this schematic drawing. The junctional complex is located at the most apical region of lateral membranes and consists of desmosomes, adherens junction and tight junctions. (b) Electron micrograph of the junctional complex in mouse intestinal epithelial cells. The tight junction is circled. (Mv, microvilli; TJ, tight junction; AJ, adherens junction; DS, Desmosome.) Scale bar, 200nm (Tsukita et al., 2001). (c) Freeze-fracture EM from the TJ region of enterocytes showing TJ strands (Schmitz et al., 2005).
These areas of apparent fusion are called ‘kissing points’. When viewed using freeze fracture electron microscopy, a technique where the membrane bilayer of cells are split open to reveal the pattern of integral membrane proteins, it becomes apparent that these kissing points actually consist of a branching network of strands that circumvent
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individual epithelial cells when observed from the apical surface (fig 1.3c; Schmitz et al.,
2005).
Hence the structure of TJs is consistent with their function as barriers. However, the seal created by TJs is not absolute and TJs are permeable to small, hydrophilic molecules and ions. In some epithelia e.g. the kidney, the TJ is an important route for ion transport (Balda and Matter, 1998).
There are two essential transport pathways across an epithelium – transcellular and paracellular i.e. through, and between, cells respectively (fig 1.4; Kondoh et al., 2006).
These two transport routes between them set up and maintain the electrochemical gradients that are required for epithelial tissue homeostasis. The transcellular pathway is managed by energy-dependent transporters and channels that are found distributed disproportionately on the basolateral and apical membranes. The paracellular pathway is formed from the essential proteins of the TJ. These span the apical intercellular space and control the passive diffusion of ions and small non-charged solutes through the paracellular space.
Whereas the transcellular transport mechanisms are well investigated, the paracellular pathway has only just begun to be elucidated (Schneeberger and Lynch, 2004).
Paracellular permeability varies widely among different epithelia and this appears to be dependent on the protein composition of the TJs in that epithelium. However, TJ permeability can also be changed as a result of physiological and pathological stimuli
(Bentzel et al., 1991). For example, a number of cytokines and growth factors can change the permeability of TJs. Thus TJs should not be viewed as simple occluding barriers rather, they are dynamic structures whose permeability can change rapidly depending on the needs of the tissue (Tsukita et al., 2001).
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Figure 1 .4 Schematic representation of the tight junction's basic structural components and the paracellular and transcellular pathways of transport across the epithelium. Transmembrane proteins claudin, occludin and junctional adhesion molecules (JAM) may interact across the intercellular space to create a semi permeable barrier. Cytoplasmic plaque provides a direct link to the actin cytoskeleton.
1.2.1 Other functions of tight junctions
Aswellastheirroleasbarriers,TJsalsopossesswhatisknownas‘fencefunction’.
This refers to the ability of TJs to prevent the free flow of plasma membrane proteins and lipids between apical and basolateral cell surfaces thus maintaining normal cell surface polarity (Van Meer et al., 1986). Specific transmembrane proteins of the TJ are also thought to be involved in the restriction of lipid diffusion (Balda and Matter, 1998).
Finally, TJs have recently been shown to include several protein complexes that not only regulate cell polarisation and junctional assembly but also control signalling molecules that
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play a crucial role in cell differentiation, proliferation and gene expression (Aijaz et al.,
2006).
1.2.2 Molecular composition of tight junctions
Many studies over the past few years have begun to identify the mechanisms that allow selective paracellular diffusion. This has provided much insight into the molecular composition of TJs (Aijaz et al., 2006). TJs are multi protein complexes consisting of transmembrane proteins, linked to the actin cytoskeleton via cytoplasmic proteins (Aijaz et al., 2006). Approximately 36 TJ proteins have been identified. Transmembrane proteins, principally claudins and occludin, are involved in creating the semi-permeable barrier whereas cytosolic proteins not only link the membrane components to the actin cytoskeleton, but also participate in signalling between the TJ and the cell nucleus. The different proteins all have unique functions which are beginning to be elucidated (Niessen,
2007) and the main components are described below.
1.2.3 Transmembrane components
a) Occludin
Occludin was the first integral membrane protein of the TJ to be identified. Furuse et al (1993) used a membrane fraction derived from the junctional complex in chick liver as an antigen in rats. This antigen produced an antibody that recognised occludin, an integral membrane protein of 65 kDa when analysed by immunoblotting.
Occludin is found in both epithelial and endothelial TJ sheets (Furuse et al., 1993) and unlike other TJ components whose expression is not necessarily restricted to the TJ
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complex, occludin is only ever found located at TJ complexes (reviewed by Niessen,
2007). For this reason, occludin is the best morphological marker of the location of TJ complexes.
Occludin has been demonstrated to be a functional constituent of TJs: its overexpression caused a rise in the trans-epithelial electrical resistance (TEER – a well established measure of TJ function; Karnaky, 1992) and the number of TJ strands in cultured MDCK cells (Madin-Darby Canine Kidney Cells) (McCarthy et al., 1996).
However, lack of occludin does not appear to be a lethal phenotype. Mice carrying a null mutation in the occludin gene were born healthy and the strands typical of TJ complexes could be observed in epithelial tissues. However, in later life these mice developed several abnormalities in different tissues, specifically chronic inflammation and hyperplasia of the gastric epithelium, deposition of minerals in the brain, testicular deterioration, loss of cytoplasmic granules in the salivary gland striated duct cells and compact bone thinning
(Saitou et al., 2000). To further understand the role of occludin in these permeability disorders, a recent study created a knock down of occludin both in vitro and in vivo in intestinal epithelial cells. This caused an increase in the flux of a macromolecular, paracellular probe across the intestinal epithelial TJ barrier (Al-Sadi et al., 2011).
However, flux of ions across the TJ was not affected suggesting that occludin is a specific barrier to macromolecular transport. Controversially, another study indicated that the barrier function of the TJ was not affected in occludin-deficient embryonic stem cells
(Saitou et al., 1998). Therefore, the physiological function of occludin in TJs is still unclear.
27
b) Claudins
The existence of TJ strands and barrier function in occludin knockout mice prompted Tsukita and his associates to look for additional protein components of TJs.
Biochemical efforts led to the discovery of the claudin family of proteins (Furuse et al.,
1998).
Claudins (clds) are a superfamily of proteins containing at least 24 homologous members in mice and humans. The expression of isoforms is tissue specific and this is thought to be responsible for the heterogeneity of paracellular characteristics between different epithelia (Tsukita and Furuse, 2002). For example, cld-3 is found largely in the lung and liver and to a small extent in the kidney, testis, gastrointestinal tract and skin.
Claudins-4, -7, and -8 are mainly expressed in the lung and kidney. Claudin-5 is expressed in all tissues (Morita et al., 1999) and cld-11 only expressed in the brain and testis (Tsukita and Furuse, 2000). This heterogeneity in expression between different epithelia is thought to be responsible for the different paracellular characteristics between individual epithelia and indeed, knockdown and over-expression studies have shown that clds define paracellular characteristics. Two classes of clds have been demonstrated: those with barrier sealing properties, expression of which reduces paracellular transport (isoforms -4, -5, -8, -
11 and -19); and those with pore forming properties (isoforms -2, -7, -10 and -15).
Expression of these tends to increase paracellular transport, sometimes for specific ions
(reviewed in Krause et al., 2008).
Claudins have a molecular mass of ~23kDa and consist of four hydrophobic transmembrane domains and two extracellular loops, whose sequences are distinct in different clds (Turksen and Troy, 2004; Schneeberger, 2003). These loops are thought to
28
interact across the paracellular space so creating the seal (fig 1.5; O’Neill and Garrod,
2011).
The claudins may also contribute to the structure of TJs as well as function - claudins have been shown to be directly involved in the formation of TJ strands and their barrier function in simple epithelia. For example, a significant increase in TJ permeability was observed when cld-4 was selectively removed from the TJ strands (Sonoda et al.,
1999). In addition, expression of clds in non-TJ-bearing cells such as fibroblasts results in the formation of TJ-like strands (Heiskala et al., 2001).
Figure 1.5 The interactions between loops of clds in the intracellular space providing a paracellular seal. Thus prevent the passage of large molecules (in red) across the paracellular space and permits only small, usually ions (shown in green) (O’Neill and Garrod, 2011).
29
The major role of clds in human physiology is also demonstrated by the growing list of human diseases where defects in clds have been shown to be causatively coupled
(Heiskala et al., 2001). For example, polymorphisms in cld-16 are associated with familial hypomagnesaemia with hypercalciuria, a condition in which the kidney fails to re-absorb calcium and magnesium; polymorphisms in clds-11 and -14 are associated with inherited deafness; cld-11 polymorphisms are associated with loss of central nervous system myelin and male sterility by loss of Sertoli cell TJ strands. There is also a human genetic equivalent of the cld-1 knock out mouse; neonatal sclerosing cholangitis associated with ichthyosis is a severe chronic condition associated with inflammation and fibrosis of the bile ducts that ultimately leads to liver failure. Although the primary problem in these people is a liver condition, there is also a severe skin phenotype associated with the condition. The skin is ichthyotic with a thicker stratum corneum. Patients also have erythema and patchy alopecia (Rabia et al., 2004).
Thus data from both in vitro and in vivo studies suggests that clds confer the major functional and possibly structural features to TJs.
1.2.4 Other transmembrane proteins
Althoughcldsandpossiblyoccludinappeartobethe‘mainplayers’intermsofTJ structure and function, two other transmembrane spanning proteins of lesser known function are also present at TJs.
In 1998, a new member of the immunoglobulin family called junctional adhesion molecule (JAM) was identified. JAM is found to be selectively concentrated at the TJs of various epithelial and endothelial cells. However, JAM can also be expressed on the
30
surface of circulating leukocytes, erythrocytes and platelets (Martin-Padura et al., 1998;
Mandell and Parkos, 2005). JAM appears to have diverse functions. Although it appears to have a role in barrier functions, it is also involve in leukocyte migration, platelet activation, angiogenesis and retrovirus binding (Mandell and Parkos, 2005).
A fourth transmembrane protein, tricellulin, has also been recently discovered
(Ikenouchi et al., 2005). Tricellulin is found concentrated at tricellular contacts in epithelial cellular sheets identified in epithelial cells of the kidney, intestine and stomach and in stratified epithelia such as the epidermis (Ikenouchi et al., 2005; Schluter et al.,
2007). Currently the role of tricellulin at TJs is largely unknown.
1.2.5 Cytosolic proteins
The TJ-associated cytosolic proteins exist as a protein ‘plaque’ that couples the membrane components to the actin cytoskeleton. Within this plaque are signalling molecules and transcription factors that signal between the TJ and the nucleus, which is important during cellular differentiation as the formation of TJs is intimately related to cell differentiation (Shen et al., 2006).
An important role of the cytosolic plaque proteins is recruitment of the transmembrane proteins and facilitating their localisation at the TJ complex. For example, the zonula occludens (ZO) family of proteins are a requirement for cld polymerisation at
TJs (Yamazaki et al., 2011).
Finally, another role of the cytoplasmic plaque proteins is to facilitate opening of
TJs to increase permeability. For example, myosin light chain kinase is a TJ associated
31
enzyme; phosphorylation of the cytoskeletal protein myosin light chains causes contraction of the protein which literally pulls the TJ open to increase permeability (Shen et al., 2006).
1.3 The role of tight junctions in the skin
Until recently, TJs were thought not to be important, or even exist, in skin.
However, at the beginning of this century, Langbein et al (2002) successfully identified
TJ-like structures in both human and mouse epidermis (Langbein et al., 2002). Moreover,
Brandner et al (2002), using electron microscopy confirmed the occurrence of structures typical of TJ kissing points in the stratum granulosum of human epidermis. They also demonstrated the expression of occludin, ZO-1 and several cld isoforms in human epidermis and cultured human keratinocytes. Compositionally related junctional structures in various layers of the epidermis were also identified using immunofluorescence microscopy but without providing any direct functional data to the purpose of these TJ proteins to the barrier role of the skin (Brandner et al., 2002).
Despite good observation of TJ structures, there were no direct evidence linking the
TJ proteins with the permeability barrier function of the skin, until the study conducted by
Furuse and his colleagues revealed a direct role of cld-1 in the epidermal barrier function of skin (Furuse et al., 2002). Furuse at al. studied the function of cld-1 in the skin by creating a knock out mouse lacking cld-1. Interestingly, the mouse died within 1 day of birth from dehydration due to severe defects in the epidermal permeability barrier even though the stratum corneum was apparently histologically normal. The epidermis of this mouse was also permeable to a low molecular weight tracer molecule which permeated the whole epidermis and into the stratum corneum when injected from the dermal side of the skin. However, in the wild type mouse, the tracer did not permeate beyond the stratum 32
granulosum. When wild type skin was co-stained for occludin, it was noted that the tracer was stopped in the granular layer at sites positive for occludin. This was the first demonstration that functional TJs exist in the granular layer of skin and that their disruption e.g. by knocking out cld-1, has a major impact on epidermal barrier function.
Subsequently, a second knock out animal has supported the essential role of TJs in epidermal barrier function. The E-cadherin knock out mouse also dies from dehydration around 7–12 h after birth. The loss of E-cadherin appears to cause changes in the localisation of clds-1 and -4 in the epidermis causing disruption of the epidermal barrier function (Tunggal et al., 2005). Turksen and Troy (2002) also provided further evidence for the role of TJs in skin barrier function. They developed a transgenic mouse overexpressing cld-6 in the suprabasal layers of the epidermis that also exhibited an increase trans-epidermal water loss that consequently caused death within 24 to 48 hours of birth. A recent study has also supported the role of TJ proteins in the permeability barrier in epidermal keratinocyte monolayer culture. Following transfer to high Ca2+ medium, epidermal keratinocytes develop a TEER which is indicative of the development of TJs.
This coincides with changes in the localization of occludin and clds-1 and -4 and their subsequent assembly into TJs (Yuki et al., 2007). Upon treatment of the keratinocyte monolayer with ochratoxin A, a toxin which specifically removes clds from TJs, TEER decreased and there was also an increase in the flux of paracellular tracer. This was shown to be concurrent with a decrease in the expression of cld-4. However a reduction of E- cadherin and loricrin was also observed, suggesting that changes at the TJ are probably not the only explanation for the reduced barrier function (Yuki et al., 2007). Nevertheless, all these findings suggest the critical contribution of TJ proteins to the formation of the permeability barrier in skin and cultured keratinocyte monlayers. More recently, a study by
33
De Benedetto et al (2011) has demonstrated the involvement of cld-1 in atopic dermatitis
(AD). They used haplotype tagging to show a genetic link between AD and cld-1 polymorphisms. Expression of cld-1 protein was observed to be reduced in the non- lesional skin of patients with AD. The authors also demonstrated that reduction of cld-1 using siRNA in human primary keratinocytes increased keratinocyte proliferation, perhaps providing an explanation for the increased thickness often observed in non lesional skin from patients with AD.
One study has also demonstrated the importance of TJs to the barrier against pathogens. Skin is one of the major targets of microbial assaults. For infection to occur bacteria have to invade the skin by passing through stratum corneum and then the living cell layers of the epidermis via either a para- or transcellular route. To clarify the role of TJ function as a barrier during bacterial cell infection, the expression of TJ proteins were investigated during infection with the skin pathogen, Staphyococcus aureus. Using a human keratinocyte cell line (HaCaT) and a porcine ex vivo skin infection model, clds-1, -
4, occludin and ZO-1 were shown to be down-regulated after S. aureus infection. This was accompanied by a decrease in TEER indicating the functional significance of these proteins. In addition, an almost complete loss of atypical PKC (Protein kinase C), which plays a key role in TJ formation, was demonstrated. The relocalisation of the TJ proteins in response to S aureus may suggest that TJs probably contribute to both inside-out barrier and outside-in barrier function of the skin (Ohnemus et al., 2007).
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1.3.1 Non-Junctional expression of tight junction proteins
Tight junction protein expression is observed throughout the epidermis. However, electron microscopy studies demonstrate that TJs are located granular layer in skin.
Furthermore, several authors have shown that the barrier to dye penetration is located within the granular layer at sites positive for occludin, the major morphological marker of
TJs (Furuse et al., 2002). Therefore, some TJ proteins may not be specifically located in TJ complexes. There is a precedent for this in other tissues; cld-1 is expressed in basal membranes in the rat epididymis (Gregory et al., 2001). Claudins-3 and -4 are expressed laterally in various regions of the intestine. Furthermore, in some epithelia, specific cld isoforms have an exclusively non-TJ location. For example, clds-3 and -5 are only found in basal membranes in gastric epithelial cells (Rahner et al., 2001). Therefore, it has been suggested that clds may have functions other than just barrier function. Given the observation of De Benedetto et al (2011) that silencing of cld-1 in keratinocytes leads to hyperproliferation, this raises the exciting possibility that clds may influence skin biology in ways as yet unidentified.
1.4 Ageing and skin
Ageing is a time-dependent progressive and obligatory event which occurs in all body tissues. However, skin is probably the organ most obviously influenced by ageing.
As well as changes in appearance, a decline in several functions has been recognised in aged skin. These include: decreased epidermal barrier function; reduced mechanical resistance; reduced sensory perception; delayed wound healing; reduced immunological reactions; altered thermoregulation and reduced vitamin D production (Table 1.1; Rabe et al., 2006).
35
Unlike other organs, skin is subject to two forms of ageing which occur concurrently.
Intrinsic ageing is time-dependent just like ageing in other tissues i.e. chronological ageing. Clinically the skin appears smooth and pale with fine wrinkles (fig 1.6; Wlaschek et al., 2001). Extrinsic ageing is due to interactions with the environment, i.e., chronic sun- exposure (termed photoageing), smoking, pollution, repetitive muscle movements
(squinting or frowning) and diverse lifestyle components such as diet, sleeping and overall health (Farage et.al, 2008). For example, a recent study indicated that histological changes just like those observed in old skin resulted from cigarette smoking (Khalaf et al., 2012).
Although all these factors may contribute to extrinsic ageing, of all the extrinsic factors which influence skin ageing, photoageing is probably the most important. Chronic exposure to solar ultraviolet radiation (UVR) appears to accelerate the ageing process
(Rabe et al., 2006). This premature ageing affects sun-exposed areas of the body such as the face, neck and dorsum of hands and forearms (Rabe et al., 2006).
Table 1.1 Components and systems of the skin: functions and changes with age. (Rabe et al., 2006).
Cell Function Change with age type/component/system
Low proliferation and differentiation (Yaar Numerous changes with and Gilchrest, 2003); age e.g., barrier function, Low cell signalling and growth factor Keratinocytes mechanical protection, response (Stanulis-Praeger and Gilchrest, cytokine production, cell 1986; Reenstra et al., 1996); signalling Low barrier function with injury (Elias and Ghadially, 2002) Low melanocyte number (Yaar and Synthesize pigment for Gilchrest, 2003); Melanocytes protection from UV Low life span and growth factor response radiation (Gilchrest et al., 1979) Reduced in number by 20%-50% (Yaar and Gilchrest, 2003); Langerhans’cells Antigen presentation Morphologic abnormalities (Yaar and Gilchrest, 2003); 36
Low cutaneous immune function (Sunderkotter et al., 1997; Sauder, 1989) Synthesis and degradation Low in number (Yaar and Gilchrest, 2003); Fibroblasts of ECM (extra cellular Low growth factor response (Edwards et matrix) al., 1996) Low biosynthesis (Uitto et al., 1989); Collagen ECM component High stability and resistance to enzymatic degradation (Bentley, 1997) Low microfibril content (Varadi, 1972); Elastin ECM component Porous, indistinct and fragmented appearance (Lewis et al., 2004) Tissue inhibitors of Protect collagen and matrix elastin from endogenous Low function (Hornebeck, 2003) metalloproteinases breakdown systems Dermal vascular bed Thermoregulation Structural loss (Yaar and Gilchrest, 2003) Thermoregulation, energy Structural loss (Gonzalez-Ulloa and Flores, Subcutaneous fat storage 1965; Hughes et al., 2004) Endocrine system— UV protection, calcium Low production (MacLaughlin and vitamin D homeostasis Holick, 1985) Improves collagen Endocrine system - content and quality, Low production (Dunn et al., 1997; Estrogen increase skin thickness, Ashcroft et al., 1997). enhance vascularisation. Low facial innervations (Besne et al., 2002); Sensation, Nervous system High truncal innervations (Young, 1991); thermoregulation. Low tolerance to cold exposure (Gosain and D Pietro, 2004). Delayed wound healing (Goukassain et al., 2000); Miscellaneous Low ability to repair DNA damage;
(Francis et al., 2004);
Low function of early population doubling
level of cDNA-1, an inhibitor of angiogenesis (Francis et al., 2004).
37
Figure 1.6 The clinical differences between sun exposed area (photoageing) and sun protected area (chronological ageing). Leathery, coarsely wrinkled, yellowish skin and reduced resilience appearance in photoaged skin in sun-exposed areas of the face and neck. A basal cell carcinoma is present on the right check. Sun-protected intrinsically aged skin of the upper chest shows a smooth pale and atrophic appearance (taken from Wlaschek et al., 2001).
Photoaged skin has a leathery appearance, with increased deep and rough wrinkle formation, irregular pigmentation, reduced recoil capacity, increased fragility of the skin and impaired wound healing. Carcinomas may also be present on photoaged skin
(Berneburg et al., 2000). As a result of the variability in sun exposure habits and the diverse capacity to block or repair the damaging effect of the sun among individuals, the rate of photoageing differs widely between individuals compared to the rate of intrinsic ageing. Generally, fair skin (Fitzpatrick Scale, I/II) seems to be more severely affected by photoageing as compared to darker skin types (Fitzpatrick scale, III-V, Yaar and Gilchrest,
38
2001b; Berneburg et al., 2000). The main features of intrinsic and extrinsic ageing are discussed below.
1.4.1 Intrinsic ageing
Intrinsic ageing results in subtle structural and functional alterations in all layers of the skin (Uitto, 1997; Yaar and Gilchrest, 2001a). The clinical signs of intrinsically aged skinusuallyappearonlyinoldage(≥70years).Theskinbecomesdry,pale,thinandfine wrinkles appear in addition to seborrheic keratoses which are associated with histological features of dermal atrophy and reduced amounts of fibrillar collagens and elastic fibres
(Montagna et al., 1989; Lavker et al., 1987).
In the epidermis, although no obvious changes are observed in the stratum corneum, keratinocyte shape and adhesive properties alter and there is a decline in the numberofmelanocytesandLangerhans’cells(Puizina-Ivic, 2008). Also, changes occur at the dermal-epidermal junction with decreased exchange of nutrients and metabolites between these two areas. In the dermis, decreases in the dermal volume and blood vessels have been observed (Puizina-Ivic, 2008). These changes can cause great morbidity to elderly people including alterations to drug permeability, decreased resistance to irritant contact dermatitis and severe xerosis (Ghadially, 1995). Recent evidence has shown that many of these age-related changes are due to reduced barrier function (Ghadially, 1995).
Xerosis (dryness) occurs due to loss of water from the skin leading to dry cracked skin especially on the legs. Sometimes cracks in the skin are deep enough to disturb dermal capillaries and bleeding can occur. Itching and rubbing can initiate an inflammatory response. Subsequently, environmental pathogens can enter the skin leading to the characteristic increase in contact dermatitis which is associated with the elderly (Tanei, 39
2009). The wound healing response is also known to be slower in aged skin which can leave the body at risk of infection (Farage et al., 2009). A factor associated with age- related changes to barrier function is a fall in the hydration of the stratum corneum. This is due to changes in the organization of lipids within the intercellular spaces of the stratum corneum such as decreases in ceramide, cholesterol and fatty acids levels. These are important in maintaining the permeability barrier to water (Rogers et al., 1996). To date, how TJs change in ageing skin has not been investigated.
1.4.2 Photoageing
Histological alterations in photoaged skin are prominent compared to those in intrinsically aged skin and both the epidermis and dermis are affected by photoageing. In the epidermis, thickening is usually observed with irregular distribution of melanocytes with a loss of rete ridges (Rigel et al., 2007). In the dermis, the degree and depth of damage depends on the amount of UVR exposure (Berneburg et al., 2000). The major histological characteristic of photoageing is solar elastosis occurring as result of accumulated dystrophic elastic fibres which are not observed in chronologically aged skin
(Mitchell, 1967; Berneburg et al., 2000). Basophilic degeneration may occur resulted from the replacement of mature collagen fibers by collagen with a distinct basophilic appearance
(Berneburg et al., 2000). Moreover, higher levels of glycosaminoglycans and fragmented elastic fibers are found in photoaged skin, with an increase in the dermal extracellular matrix proteins such as elastin, glycosaminoglycans and interstitial collagen (Berneburg et al., 2000). Low levels of collagen I and III are also found in the dermis of photoaged skin
(Talwar et al., 1995) along with less anchoring fibrils at the dermal-epidermal junction
(Craven et al., 1997). Therefore, severely photodamaged skin has significantly impaired 40
elasticity and the skin loses the ability to return to its original state after distortion due to alterations in the organisation of collagen and elastin (Baran and Maibach, 2004). Beside clinical features, there are several methods that can measure and quantify photoageing include histopathological evaluation and Skin microtopography (Wurm et al., 2012). Also a nine-point photonumeric standard scale shows an accurate assessment of photoageing by using photographs of subjects representing grades of photodamage from none to severe
(Griffiths et al., 1992). Several factors may contribute to the severity of photoaged skin include age, sex, geographic location, and skin phototype (Malvy et al., 1999).
Much of the damage caused by UVR is due to the production of reactive oxygen species (ROS). Data from in vitro and in vivo studies have shown that upon exposure to
UVR, cutaneous tissue generates free radicals and other types of ROS. Transient or permanent changes in genomic and proteomic function and structure are caused by the elevated levels of ROS generated in dermal as well as epidermal cells causing local inflammation and alteration to signal transduction pathways involved in cell growth, differentiation and degradation of connective tissue (Yaar and Gilchrest, 2001a;
Scharffetter-Kochanek et al., 1997; Masaki et al., 1995)
In human skin, UVR activates the mitogen-activated protein kinase (MAPK) pathway which in turn induces activator protein 1 (AP-1). AP-1 regulates matrix metalloproteinase (MMP) gene expression which has significant down-stream effects on dermal extracellular matrix (ECM) remodelling by degrading the matrix proteins. The
MMP genes induced by ROS increase the degradation of collagen and the elastic tissue components by inactivating the naturally occurring tissue inhibitors of metalloproteinases
(TIMPs) (fig 1.7) (Trautinger, 2001; Fisher et al., 1997; Svobodova et al., 2006). These
41
data explain at least partially, the damage to the extracellular matrix observed in photoaged skin.
Figure 1.7 Hypothetical model of the dermal damage and photoageing caused by UV radiation. Matrix metalloproteinases (MMPs) in keratinocytes (KC) in the outer layers of skin, as well as fibroblasts (FB) in connective tissue are activated in response to UV radiation. These enzymes degrade collagen in the extracellular matrix of the dermis depending on the activity of tissue inhibitor of matrix metalloproteinases-1 (TIMP-1). The organization and/or composition of ECM is affected by imperfect repair of tissue leaving accumulated altered matrix (solar scar) and finally, observable photoageing (wrinkles) (taken from Fisher et al., 1997).
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1.4.3 Acute effects of ultraviolet radiation on skin
Based upon wavelength, UVR emitted from the sun is divided into three categories:
UVA (320-400 nm); UVB (290-320 nm) and UVC (200-290 nm) (Beissert and Granstein,
1995). In contrast to UVA and UVB, UVC does not reach the earth in measurable amounts because it absorbed by the ozone layer in the atmosphere. Thus, all the biologically important effects of UVR are mediated mainly by UVA and UVB. Generally the penetration of UVR into the skin depends on the wavelength. Whereas UVA penetrates both the epidermis and the dermis, UVB penetrates only the epidermis (Beissert and
Granstein, 1995).
UVR is mostly absorbed by skin chromophores. These are molecules with the ability to absorb photons of light which in turn causes electrons within the chromophore to move to a more excited state. Chromophores in skin include DNA and urocanic acid
(Wondrak et al., 2006).
a) Erythema (Sunburn)
Acute exposure to UVR causes visible skin changes such as sunburn, whereas chronic exposure results in photoageing and cancer. Erythemal response of acute UVR exposure varies between individuals depending on their skin phototype, UVR dose and emission spectrum; it reaches a peak at 24 hours and persisting usually for no longer than
72 hours (Epstein, 1970; Young, 2006). The sensitivity of sunburn between individuals is usually determined by visual assessment of MED. In the dermis, molecular and cellular alterations that include the appearance of inflammatory cells (Hawk et al., 1988; Gilchrest et al., 1983) and in the epidermis p53 (Burren et al., 1998) and apoptotic sunburn cells
43
(Sheehan and Young, 2002) are found as a result of acute UVR exposure (reviewed in
Battie and Verschoore, 2012).
b) Melanogenesis
Dendritic mealnocytes produce melanin in response to UVR. To protect DNA from the UVR damage, melaninrepeatedlyaccumulatesasanuclear‘‘cap’’insidekeratinocytes or melanocytes (Whiteman et al., 1999). Melanocytes number found to be declined with age (Yaar and Gilchrest, 2003); along with a low life span and growth factor response
(Gilchrest et al., 1979).
c) Immune effects
Epidermal Langerhans cells protect the skin against any infectious agent. However,
Acute exposure to UVR induce immunosuppression caused by a reduction in Langerhans cells number (Yaar and Gilchrest, 2003), morphologic abnormalities (Yaar and Gilchrest,
2003), and low cutaneous immune function (Sunderkotter et al., 1997; Sauder, 1989).
1.5 Epidermal barrier function and Ultraviolet radiation
Ultraviolet radiation also plays an important role in disturbing epidermal barrier function. For example, irradiation of hairless mice with acute UVB at 1.5-7.5 times the minimal erythemal dose (MED) increased trans-epidermal water loss (TEWL) in a time- and dose-dependent manner suggesting that UVB modulates barrier function. Increased proliferation of keratinocytes was also observed in these mice (Haratake at al., 1997). A
44
further mouse study (Rodriguez-Martin et al., 2011) has also demonstrated that an acute dose of 5xMED also increases permeability of mouse skin.
There has also been a study on the effects of UVR on the human permeability barrier (Lim et al., 2007). In this study, healthy Korean men were given a dose of 0.5-
2xMED on the back and TEWL was monitored for 28 days post-irradiation. Although both low and high doses resulted in an increase in TEWL from day 1 post-irradiation, skin irradiated with low doses of UVR (0.5 and 0.75 MED) recovered within 3-4 days, whereas skin irradiated with higher doses required longer for TEWL to return to pre-irradiation levels. These data show that the skin permeability barrier is perturbed by UVR but can recover with time. However, in general the mechanisms underlying perturbation of the barrier have not been investigated. One study in keratinocytes has suggested one potential mechanism: down-regulation of aquaporin-3 (AQP3) is induced by UVR exposure in primary human keratinocytes. This was accompanied by an increase in water permeability and induced dehydration in the keratinocytes (Cao et al., 2007).
Thus, from these studies, it is clear that UVR perturbs the epidermal barrier.
However, currently little is known regarding the involvement of TJs in barrier perturbation. A recent study suggested that TJ protein expression in hairless mice was perturbed following UVB irradiation. An alteration of TJ-related molecule localisation occurred following UVB exposure and this was accompanied by an increase in dye penetration through the TJ suggesting that its barrier function was compromised.
Moreover, cld-1 and occludin expression were increased after 3-4 days of UVB irradiation and stayed elevated until day 10 when epidermal barrier recovery occurred (Yamamoto et al., 2008). Another study showed a deterioration of TJ function after UVB irradiation of human skin xenografted onto mice. This study also investigated the effects of UVB 45
irradiation of human skin equivalents (HSE) and in vitro cultured keratinocytes (Yuki et al., 2011). TEER measurements taken after 48h of calcium-induced formation of TJ in cultured keratinocytes demonstrated a decrease in barrier function in a dose dependent manner. This was associated with a discontinuous membranous pattern of TJ proteins
(occludin, clds-1 and -4) observed by immunofluorescence (Yuki et al., 2011). Thus, from these preliminary studies in model systems, TJs appear to be modulated by UVR.
However, how they may change in humans due to acute or chronic UVR exposure
(photoageing) has not been studied.
1.6 The role of tight junctions in age related changes
One theory of ageing suggests that systemic inflammation, as a result of life-long burden of infectious diseases, is a major determinant of life span (Crimmins et al, 2006,
Finch et al., 2007). These led Skrovanek et al (2007) to postulate that leak through epithelial barriers may underlie the increase in inflammation that is often associated with old age. Several studies point to an increase in the circulating levels of pro-inflammatory cytokines and C-reactive protein in the elderly (Mascarucci et al., 2001, Salvioli et al.,
2006, Nilsson, 1968) and Skrovanek et al hypothesise that increased leakiness in epithelial barriers might lead to an increase in the ingress of immunostimulatory agents such as toxins and bacteria. Translocation of potential antigens is observed in acute trauma to a barrier e.g. in inflammatory bowel disease, but Skrovanek et al (2007) postulate that a chronic low grade leak though epithelial barriers may also occur during ageing. The same authors have also shown a correlation between calorie restriction – a well known mechanism of increasing longevity (Orentreich et al., 1993; Zimmerman et al., 2003) and strengthening of epithelial barriers. Using a renal epithelial cell line they showed that
46
restriction of sulphur containing amino acids in the cell medium resulted in changes to cld expression that increased barrier function as measured by TEER.
The role of TJs in age-related changes is currently an under investigated area. This is surprising given the role of TJs as barriers, with crucial roles in normal homeostasis.
However, limited work, primarily using rodent models, has highlighted increases in paracellular permeability i.e. reduced barrier function in the gastrointestinal tract (Mullin et al., 2002), the lung (Tankersley et al., 2003), the blood brain barrier (Mooradian et al.,
2003) and the epididymis (Levy et al., 1999) with increasing age. In these studies, although increased leakiness of epithelial barriers was observed in aged rodents compared to young, in general the molecular mechanisms underlying these observations were not investigated. However, more recently age-related changes in the expression of clds in mouseliver,kidney andpancreashavebeendemonstrated(D’Souza et al., 2009). They investigated the expression levels of clds-1, -2, -3, -4, -5 and -7 and showed that in general, the levels of these proteins decreased in ageing in all tissues studied. These age-related changes in TJ protein expression may explain the decrease in epithelial barrier function observed in these tissues in ageing. Thus, although a few studies in rodents have identified increases in epithelial permeability and changes in cld expression, currently there are no studies in human tissues investigating how TJ protein expression changes in ageing.
Additionally, skin TJs have not been studied to date in any system.
Hypothesis, aims and objectives:
The epidermis of skin provides a critical barrier which protects the body from dehydration and infection by bacteria. This barrier is known to alter with intrinsic ageing leading to xerosis, increases in contact dermatitis and delayed restoration following 47
wounding. Additionally, UVR is known to modify epidermal barrier function and limited evidence from model systems suggests that TJs, critical components of the epidermal barrier, may be modified by UVR. Currently it is not known how TJs alter with age either in intrinsic or photoageing, nor how any changes might impact upon skin. The hypothesis underlying this work is that:
The decrease in barrier function observed in ageing in skin is due to modification in
TJ structure or function.
To date all the data pertaining to decreased TJ barrier function in ageing have come from rodent models. To the best of my knowledge, no human studies have been conducted to ascertain how TJs might alter in ageing. Therefore, the aims of this study are, using human tissue:
1) To characterise changes in TJ protein expression in young (18-30 years) versus old (65-
75 years) skin;
2) To characterise changes in TJ protein expression in photoaged versus photoprotected skin;
3) To study the acute effects of UVR on TJ protein expression in photoprotected skin.
Using cell lines:
4) To investigate the potential mechanisms underlying any TJ-associated changes observed.
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2. MATERIAL AND METHODS
2.1 Materials
Primary antibodies were purchased from Invitrogen (Paisley, UK). These were: rabbit anti-claudin-1 (JAY.8); mouse anti-claudin-4 (3E2C1); rabbit anti-claudin-5
(Z43.JK); mouse anti-claudin-7 (5D10F3); rabbit anti-claudin-8 (ZMD.446); rabbit anti- claudin-12 (ZMD.398); mouse anti-ZO-1 (ZO1-1A12) and mouse anti-occludin (OC-
3F10). Secondary antibodies (Jackson Immunoresearch Labs, INC.; West Grove, PA,
USA) used to visualise immunostaining were fluorescein isothiocyanate (FITC)- conjugated goat anti-rabbit IgG (H+L) and goat anti-mouse IgG (H+L). The secondary antibody used for Western blotting was horseradish peroxidase (HRP-) conjugated goat anti-rabbit IgG (BioRad; Hercules, CA, USA). TaqMan probes and primers were purchased from Applied Biosystems (Warrington, UK). Unless otherwise stated, all other reagents were purchased from Sigma Aldrich Ltd. (Poole, UK).
2.2 Patient Samples
Studies were performed according to the Declaration of Helsinki 2009. Ethical approval was granted by North Manchester Local Research Ethics Committee (ref:
09/H1006/23), and written informed consent was obtained from each volunteer.
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2.2.1 Intrinsic ageing study
For the intrinsic ageing study, two cohorts of healthy Caucasian volunteers were recruited: Cohort 1, age range: 18-30 years (i.e. young cohort); Cohort 2, age range: 65-75
(aged cohort). Both cohorts had n=8 volunteers each. Six millimetre diameter punch biopsies were taken under local anaesthesia from photoprotected buttock skin. This was carried out by Gill Aarons, a research nurse within the Dermatopharmacology Unit
(Salford Royal Teaching Hospital). The biopsies were embedded in Optical Cutting
Temperature (OCT) embedding matrix (Miles, IN, USA) and stored at -80oC until required.
2.2.2 Photoageing study
For the photoageing study, skin was taken from 3 distinct anatomical sites from the
‘agedcohort’(65-75 years; n=8) described above: buttock skin was used a photoprotected site whereas skin from the forearm was taken as representative photoaged site. Skin was also taken from the upper inner arm as an anatomical site control to ensure that any differences found between the forearm (photoexposed area) and hip (photoprotected area) was due to photoexposure and not to anatomical site differences. Punch biopsies (6mm diameter) were taken from each site, embedded in OCT compound and stored at -80oC until required.
2.2.3 Acute UV irradiation of human subjects
This study used archival material that was originally generated from the
‘SUNALL’studyconductedpreviously within the Photobiology Unit of the University of
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Manchester (LREC ref: #024/ST/138; Salford and Trafford Research Ethics Committee).
Written, informed consent was obtained from all patients.
Caucasian volunteers were recruited to the study, age range (33-58) years; (n=8).
The minimal erythema dose (MED) of each volunteer was established; MED testing on buttock skin was performed using a bank of Philips TL-12 tubes and neutral density grids.
This source of UVB, although it does not have the same spectrum as solar irradiation, is a standard UVB broad band lamp that has been used in many photobiology and photodermatology studies. For example, this UVB source caused a reduction in the migration of Langerhans cells caused by a depletion in cytokines in PLE (Polymorphous light eruption) patients (Kolgen et al., 2002 and Kolgen et al., 2004). The irradiance of the bank of TL-12 tubes was measured using a UVX radiometer equipped with a UVX-31 detector (Ultra-Violet Products Upland, CA, USA) calibrated for the source using a double grating spectrophotometer and standards traceable to the UK National Physical Laboratory.
A single 1cm2 area of buttock was exposed to 200mJ/cm2 of UVB Philips (TL-12).
At 4 and 24 hours following UV exposure, a 6mm diameter punch biopsy was taken under local anaesthesia (carried out by research nurse Margaret Brownrigg) from the 1cm2 sites previously exposed to UVB. A control biopsy was taken from the contra lateral buttock which had not been exposed to UVB radiation (0h). Biopsies were stored at -80°C embedded in OCT compound until required.
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2.3 Histology
2.3.1 Sectioning of tissue
Skin tissue blocks were mounted onto a cryostat chuck (OFT Cryostat; Bright
Instruments, Cambridge, UK). Tissue was maintained at -20°C in the cryostat and coarsely sectioned (30µm sections) until the surface of the tissue was exposed. Subsequently, the thickness was decreased and 10µm sections were cut. Three sections were mounted onto each subbed slide (appendix 1). Sections were labelled and stored in boxes at -20°C.
2.4 Immunostaining of human skin
Frozen sections were air-dried at room temperature. Sections were then fixed accordingly to provide optimal staining for each primary antibody. For cld-1, the sections were fixed using a methanol : acetone mix (50:50 v/v) for 10 minutes at -20°C. Sections to be stained for clds-5, -7 and -8, occludin and ZO-1 were fixed with 100% acetone for 10 minutes at -20°C; cld-4 stained sections were fixed with 4% (v/v) paraformaldehyde (PFA, see appendix 2) for 10 minutes at room temperature (Table 2.1). Claudin-12 sections were fixed with 100% methanol for 5 minutes at -20°C followed by 100% acetone for 3 minutes at -20°C (a summary of the fixative methods used is found in Table 2.1). Sections were then hydrated in Tris-buffered saline solution (TBS; appendix 2) for 60 minutes at room temperature.
Cell membranes were permeabilised using 0.5% (v/v) Triton-X100 (appendix 2) in
TBS (10 minutes at room temperature) and the sections were again washed (3x 5 minutes;
TBS). Blocking solution containing normal serum and bovine serum albumin (BSA)
(appendix 2) at a concentration appropriate for the primary antibody (as summarised in
52
Table 2.1) was applied to each section for 1 hour at room temperature to minimise any non-specific background staining. The primary antibody was then applied at an appropriate dilution (see Table 2.1). The sections were then incubated overnight at 4°C. Sections to be used as negative controls were incubated in blocking solution containing no primary antibody.
Sections were subsequently washed (2x 5 minutes; TBS) and secondary FITC- conjugated antibody applied (Table 2.1). Sections were incubated for 1 hour at room temperature in the dark. They were then washed (3x 5 minutes; TBS) before mounting using VectaMount™ (permanent mounting medium; Vector Laboratories, Inc.;
Burlingame, CA, USA).
Sections were observed using a Keyence BioZero fluorescent microscope
(Keyence; Tokyo, Japan) and images captured. Images of three sections from each anatomical site were captured using the image capture function on the microscope. These images were saved as TIFF files and analysed using Image J software (NIH; Bethesda,
MD, USA). For clds-1, -4, -5 and -12 which are expressed throughout the epidermis an area of staining was selected from stratum corneum to dermal-epidermal junction (DEJ) using a free hand selection tool from the status bar in Image J. The intensity of the fluorescent signal in the selected area was then measured and the mean of the intensity calculated (fig 2.1a). Claudins-7 and -8, occludin and ZO-1 are expressed specifically in the granular layer of the epidermis. For these, images were saved as bitmaps (BMP) and a line was drawn from the SC to the end point of the TJ protein expression. Then a plot profile was measured for each line representing the fluorescent intensity (fig 2.1b).
StatisticalsignificancewasestablishedusingStudent’st-test or ANOVA, as appropriate.
53
Significance was taken at the 95% confidence level. In the results sections, data are expressed as mean ± standard error of the mean.
Table 2.1 Fixation methods of tight junction proteins and dilutions of primary and secondary antibodies used in immunostaining.
Dilution of Secondary % of Dilution of Primary Fixation Fluorescein Fixation method block primary antibodies time isothiocyantae (FITC)- solution antibody conjugated antibody MeOH:Acetone Rabbit anti- 10 (50:50 v/v) 1% block 1:100 1:160 claudin-1 minutes -20°C Mouse anti- 4% (v/v) PFA 10 1% block 1:50 1:400 claudin-4 RT minutes
Rabbit anti- 100% acetone 10 3% block 1:50 1:200 claudin-5 -20°C minutes
Mouse anti- 100% acetone 10 3% block 1:50 1:200 claudin-7 -20°C minutes Rabbit anti- 100% acetone 10 1:100 3% block 1:50 claudin-8 -20°C minutes 100% Methanol Rabbit anti- -20°C followed by 5 minutes 1:200 3% block 1:50 claudin-12 100% acetone - 3 minutes 20°C. Mouse anti- 100% acetone 10 3% block 1:50 1:200 ZO-1 -20°C minutes Mouse anti- 100% acetone 10 3% block 1:50 1:200 occludin -20°C minutes
54
Figure 2.1 Diagram of immunofluorescent staining intensity measurement using ImageJ software. (a) Shows an area measurement of the immunofluorescent staining of cld-1 antibody throughout the epidermis. (b) Shows a line measurement of the immunofluorescent staining of cld-7 antibody from the stratum corneum to the stratum basale.
2.4 Cell culture
Normal Human epidermal keratinocytes (NHEK) were obtained from PromoCell
(Heidelberg, Germany) as frozen cultures in cryovials.
55
2.4.1 Recovering cells from liquid nitrogen
To recover the cells, the samples were rapidly warmed to 37oC in a water bath with gentle agitation immediately following removal from liquid nitrogen. Then they were placed into 25ml of growth medium in a 75 cm2 flask (Fisher, Loughborough, UK).
Cells were maintained in keratinocyte basal medium (KBM; PromoCell) containing supplement mix (bovine pituitary extract 0.004mg/ml, epidermal growth factor
(recombinant human) 0.125ng/ml, insulin (recombinant human) 5µg/ml, hydrocortisone
0.33µg/ml, epinephrine 0.39µg/ml and transferrin, holo (human) 10µg/ml) and 0.06mM
CaCl2 (PromoCell). Medium was substituted twice weekly and cells of passages 2-6 were
2 o cultured in 75cm T-flasks (Fisher; Loughborough, UK) at 37 C in a 5% CO2 constant humidity environment. Monolayers were sub-cultured once grown to 80% confluency using 2ml of 0.04% trypsin/0.03% EDTA (PromoCell). Then 2ml of trypsin neutraliser solution (PromoCell) was added once the cells were detached. The cell suspension was centrifuged for 3 minutes at 220 xg. The supernatant was discarded and the cells were resuspended in KBM. Cells were then seeded into 75cm2 flasks containing keratinocyte medium at approximately 5 x 103 cells/cm2 which was established by counting the cells using a Z1 Beckman Coulter cell counter (High Wycombe, UK).
2.5 Ultraviolet irradiation of cells
NHEK were grown in 6-well plate until they were 95% confluent. Culture medium was removed and cells were washed with phosphate-buffered saline (PBS). The lid of the plate was removed and the cells were irradiated in 0.5ml of PBS using a UV irradiation source. This was a single 20W Phillips TL-12 fluorescent tube emitting 280-400nm (peak
313nm) (fig 2.2). Source irradiance was monitored using a UPX radiometer and 313nm 56
detector (UV Products, California) calibrated for the source using a Bentham double grating spectrophotometer and a deuterium standard lamp supplied by the National
Physical Laboratory (Middlesex, UK). Irradiation doses were calculated according to the following formula:
Dose (mJ/cm2) = Irradiance (mW/cm2) x Time (s)
All doses referred to in this report are for the UVB (280-315nm) output of the TL-
12 source. Cells were exposed to different UV doses (0, 5, 10, 15 and 20 mJ/cm²). After irradiation, PBS was replaced with fresh culture medium and the cells were incubated for
24 hours.
Figure 2.2 Relative spectral output of TL-12 source.
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2.6 Measurement of cell viability
2.6.1 MTT assay
MTT (3-[4, 5-dimethylthiazol- 2-yl)]-2,5-diphenyl tetrazolium bromide) was used to measure cell viability after irradiation. Medium containing 10% (v/v) MTT stock solution (5mg/ml) in PBS was added to each well and samples were incubated for 4 hours at 37oC. Medium was then removed and replaced by 500µl of dimethyl sulphoxide
(DMSO). The plates were shaken for 15 minutes to dissolve the formazan product and the absorbance of each well was measured at a wavelength of 540nm using a Labsystems multiscan® MC spectrophotometer (LabSystems; Finland). Experiments were performed in triplicate.
2.7 Measurement of Trans-epithelial Electrical Resistance (TEER)
TEER (ohms.cm²) was measured using an Evometer (World Precsion Instruments;
Herts, UK) fitted with 'chopstick' electrodes. NHEK cells were grown on Transwell™ polycaronate microporous filters with a pore size of 0.4m (Corning Costar;Fisher,
Loughborough, UK) before performing the experiment. All experiments were performed in duplicate with n=6.
2.8 Preparation of protein extracts
NHEK grown to confluence in a 6-well plate were washed twice with ice-cold
PBS. After removing the PBS, 100l ice-cold extraction buffer (NaCl (120mM), HEPES
(25mM, pH 7.5), Triton-X100 (1%v/v), NaF (25mM), NaVO4 (1mM), SDS (0.2% w/v), aprotinin (10g/ml), leupeptin (10g/ml), PMSF (100M) and pepstatin (5g/ml)) was
58
added to each well of the 6-well plate. Cells were scraped and the samples removed to
1.5ml Eppendorf® tubes. A further 100µl of buffer was then added to each well and the remaining cells scraped and placed into the same Eppendorf® tube. The sample was incubated on ice for 30 minutes and inverted every so often to resuspend the pellet. The sample was centrifuged (MSE, UK) for 15 minutes at 13000 xg to sediment debris. The supernatant containing the protein extract was removed into a fresh tube and the protein concentration determined (see section 2.10). All supernatants were stored at -20oC for further experimental analysis.
2.9 Determination of protein concentration
The protein concentration of samples was determined using a Bicinchoninic Acid
(BCA) assay kit according to the manufacturer's instructions (Pierce Biotechnology;
Rockford, UK).
Briefly, a standard curve was designed using BSA as the standard (Table 2.2).
Samples were diluted 1:5 with diluent for the assay and absorbance readings were measured at a wavelength of 562nm, using a spectrophotometer (Labsystems multiscan®
MC, Labsystems; Helsinki, Finland). An example of a standard curve is shown in fig 2.3.
59
Table 2.2 Standard curve standards. A standard curve for the BCA protein assay composed of 7 standards.
Volume of Protein concentration of standard Volume of BSA (l) from a 2mg/ml diluents (mg/ml) stock solution (l distil H2O)
0.0 0.0 100
0.25 12.5 81.5
0.50 25.0 75.0
0.75 37.5 62.5
1.00 50.0 50.0
1.50 75.0 25.0
2.00 100 0.0
1.6 y = 0.7425x 1.4
1.2
1
0.8
0.6 Absorbancce at 562 nm 562 at Absorbancce 0.4
0.2
0 0 0.5 1 1.5 2 2.5 Protein concentration (mg/ml)
Figure 2.3 BCA Standard curve. Example of a typical standard curve for the BCA protein assay.
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2.10 SDS-PAGE (Sodium dodecyl sulfate polyacrylamide gel electrophoresis) and
Western blotting
Protein samples were loaded in equal amounts onto 12% acrylamide gels and the proteins were electrophoretically separated for 1 hour at 150mV. Proteins were then electrophoretically transferred onto PVDF membranes at 100mV for Western blot analysis.
SDS-PAGE broad range molecular weight markers were also applied to the gels to assess molecular weight (BioRad; Hertfordshire, UK). Following transfer, membranes were blocked in a 5% (w/v) skimmed milk solution (in TBS) (appendix 3) overnight at 4oC and then incubated with the primary antibody for 1 hour at room temperature in the case of cld-
1. For cld-4, occludin and ZO-1, membranes were blocked in a 5% (w/v) skimmed milk solution (in TBS) for 1 hour at room temperature and then incubated with the primary antibody overnight at 4oC. All the antibodies were used at a concentration of 1:1000 in 5% milk. Membranes were washed for 3x 5minutes in TBS containing 5% skimmed milk and then incubated with secondary antibody (HRP-conjugated goat anti-rabbit IgG) at 1:5000 dilution for 20 minutes at room temperature. Following a further 3 washes in TBS, membranes were visualised using enhanced chemiluminescence (ECL; Amersham, UK) accordingtothemanufacturer’sinstructions.Densitometrywascarriedoutasdescribedin
McLaughlin et al., 2004 (see appendix 3 for buffer recipes).
2.11 Immunofluorescent staining of keratinocytes
Cells were cultured in 8-well Lab-Tek chamber slides (Nunc International) and irradiated with UV doses of 0 and 15 mJ/cm². Then, the cells were fixed with methanol : acetone (50:50 v/v) for 10 minutes at -20°C for cld-1 and 100% methanol for 10 minutes at
-20°C for cld-4. Occludin and ZO-1 were both fixed 4% (v/v) paraformaldehyde for 10
61
minutes at -20°C. Cells were washed with PBS solution 3 times for 5 minutes and incubated with blocking buffer [10% normal goat serum, 2% Triton X-100, 0.5% Tween
20(v/v) in PBS (containing (0.01mM NaN3] for 30 minutes at room temperature, followed by washing with PBS and incubation with primary antibodies (1:50) dilution for 1 hour at room temperature. Cells were then washed again (3x 5 minutes; PBS), and incubated with the appropriate secondary antibodies ((FITC)-conjugated goat anti-bodies) for 1 hour in the dark. Nuclei were counterstained using DAPI (4',6-diamidino-2-phenylindole) nuclear stain then washed (3x 5 minutes; PBS) before mounting using VectaMount™(permanent mounting medium; Vector Laboratories, Inc.; Burlingame, CA, USA). Cells were observed using a Keyence BioZero fluorescent microscope (Keyence; Tokyo, Japan) and images captured.
2.12 Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR)
Total RNA was extracted from cells or human tissues according to the manufacturer’s instructions using Trizol reagent (Invitrogen, Life Technologies Ltd;
Paisley, UK). Following this 3µg of total RNA was then reverse transcribed using the cloned AMV first strand cDNA synthesis kit (Invitrogen,). The mRNA levels of clds-1, -7, and -12, were examined using pre-developed Taqman assays (Applied Biosystems;
Weiterstadt, Germany) using the Step One Plus PCR system (Applied Biosystems).
Glyceraldehyde-3- phosphate dehydrogenase (GAPDH) was amplified from the same
RNA samples and served as the internal control.
62
2.13 Data presentation and statistical analysis.
In each experimental series, at least three separate experiments were performed.
Data are presented as means ± standard error. Differences were considered statistically significant at the 95% confidence level with P values calculated by the analysis of variance
(ANOVAs)testforcomparisonsonmorethan2samplesandStudent’sunpairedt-test for comparisons on 2 samples.
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3. A COMPARISON OF TIGHT JUNCTION PROTEIN EXPRESSION
IN INTRINSICALLY AGED VS YOUNG HUMAN SKIN
3.1 Introduction
As outlined in Chapter 1, human skin barrier function in some studies has been shown to decrease with age leading to such things as xerosis and decreased resistance to infection.Tightjunctionsareknowntobeessentialcomponentsoftheskin’sbarrierand animal studies in other epithelia have shown changes to TJ function and protein expression in ageing. Specifically, one study in mice(D’Souzaet al., 2009) observed reduction in the expression of clds in epithelia from tissues such as the intestine and pancreas. These changes might underlie the decrease in barrier function observed in these tissues in ageing
(D'Souza et al., 2009).
An Affymetrix® based study conducted by Lener et al (2006) compared gene expression in young vs aged individuals. One of the transcripts observed to change in young vs aged skin was cld-8 which was increased in the skin of aged individuals compared to young. Furthermore, an ‘in house’ Affymetrix® study also comparing transcripts in young vs aged individuals was also carried out by Dr Abigail Langton
(Dermatopharmacology Unit, University of Manchester; Langton et al., 2012) Therefore, an aim of this study was to analyse the Afftymetrix® data generated by Langton et al to establish which TJ gene transcripts are present in skin and whether they change with increasing age. However, to date, no study in the literature has studied TJ protein expression in skin during ageing. Moreover, to my knowledge, there is no published study of how TJ protein expression changes in ageing in any human tissue. This is perhaps understandable given the ethical constraints of working with human subjects. Therefore, 64
the primary aim of the study outlined in this chapter was to quantify TJ protein expression in aged vs. young human skin.
3.2 Results
3.2.1 Analysis of Affymetrix® data
An Affymetrix® genome array comparing gene expression in aged vs young individuals was carried out by Dr. Abigail Langton (Research Associate, School of
Translational Medicine; University of Manchester). Analysis of this array for TJ-associated proteins showed a significant increase of cld-8 and cld-11 expression in aged skin (Table
3.1). This result is consistent with the previous gene expression study conducted in aged vs. young skin (Lener et al., 2006). However in this study, only cld-8 was up-regulated in aged vs young skin. The Affymetrix® array showed no significant differences in other TJ genes in aged vs. young human skin.
Given that two independent Affymetrix® studies indicated an increase in cld-8 transcript, and the possibility that changes in other TJ proteins could also be post transcriptional, methods were developed with which to analyse TJ protein expression in skin which could then be used to compare expression in aged vs. young skin.
65
® Table 3 .1 Affymetrix genome array of TJ (modified from Langton et al., 2012).
Affymetrix fc_(Mn_old/Mn Gene Gene description p.value ® ID _young)
222549_at CLDN1 claudin 1 -1.1084 0.534385777
218182_s_at CLDN1 claudin 1 -1.1435 0.580775292
222549_at CLDN10 claudin 10 1.09871 0.181596055
218182_s_at CLDN10 claudin 10 1.33562 0.270832422
222549_at CLDN11 claudin 11 1.85233 0.031628244*
218182_s_at CLDN11 claudin 11 -1.1241 0.179901693
222549_at CLDN12 claudin 12 1.03548 0.74880225
218182_s_at CLDN14 claudin 14 1.07026 0.365493849
222549_at CLDN15 claudin 15 1.0932 0.573678985
218182_s_at CLDN15 claudin 15 1.00471 0.969067764
222549_at CLDN16 claudin 16 -1.1652 0.113912898
218182_s_at CLDN17 claudin 17 1.01914 0.751497717
222549_at CLDN18 claudin 18 -1.0698 0.311000318
218182_s_at CLDN18 claudin 18 1.0751 0.435718961
222549_at CLDN18 claudin 18 -1.0311 0.680316867
218182_s_at CLDN18 claudin 18 -1.0074 0.918448284
222549_at CLDN19 claudin 19 -1.1284 0.095683838
218182_s_at CLDN19 claudin 19 -1.1011 0.199929955
222549_at CLDN19 claudin 19 -1.0159 0.792146112
218182_s_at CLDN2 claudin 2 -1.0646 0.345423067
222549_at CLDN20 claudin 20 -1.1235 0.315558741
218182_s_at CLDN23 claudin 23 1.54878 0.154758513
222549_at CLDN23 claudin 23 1.16648 0.406517074
218182_s_at CLDN23 claudin 23 1.0516 0.481715952
66
222549_at CLDN3 claudin 3 -1.1164 0.158687501
218182_s_at CLDN3 claudin 3 1.0197 0.83881659
222549_at CLDN4 claudin 4 1.07612 0.414766108
218182_s_at CLDN4 claudin 4 -1.1131 0.546083302
222549_at CLDN5 claudin 5 1.14384 0.305135294
218182_s_at CLDN6 claudin 6 1.01437 0.816146959
222549_at CLDN6 claudin 6 1.0176 0.861262975
218182_s_at CLDN7 claudin 7 1.30229 0.124694582
222549_at CLDN8 claudin 8 1.33031 0.015652697*
218182_s_at CLDN9 claudin 9 1.11686 0.102806424
CLDND claudin domain 208925_at -1.2586 0.130615528 1 containing 1
CLDND claudin domain 1554149_at -1.0765 0.664656209 1 containing 1
CLDND claudin domain 239146_at 1.04393 0.81259698 1 containing 1
CLDND claudin domain 231162_at 1.08836 0.206610715 2 containing 2
209925_at OCLN Occludin -1.0214 0.914535477
3.2.2 Detection and quantitation of tight junction proteins using immunofluorescent
staining
Method Development
Initial experiments assessed the effect of different fixative methods and dilutions of primary and secondary antibodies (see Table 2.1 in section 2.4) to optimise the immunostaining of TJ proteins clds-1, -4, -5, -7, -8 and -12, ZO-1 and occludin in photoprotected skin from hip or upper inner arm (fig 3.1a, b, c, d, e, f, g and h shows TJ
67
protein staining during various stages of optimisation). These particular proteins were studied because, with the exception of cld-8, their expression has already been demonstrated in rodent skin or keratinocytes (Furuse et al., 2002; Tebbe et al., 2002;
Mooradian eta al., 2003). Claudin-8 is of interest because as mentioned previously, an up- regulation in its gene expression was found in aged vs young skin in two independent
Affymetrix® studies (Lener et al., 2006; Langton et al., 2012).
68
Figure 3 .1a The optimisation method of the immunostaining of cld-1 TJ protein in human buttock epidermis (Magnification 20x). 69
Figure 3 .1b The optimisation method of the immunostaining of cld-4 TJ protein in human buttock epidermis (Magnification 20x). 70
Figure 3 .1c The optimisation method of the immunostaining of cld-5 TJ protein in human buttock epidermis (Magnification 20x). 71
Figure 3 .1d The optimisation method of the immunostaining of cld-7 TJ protein in human buttock epidermis (Magnification 20x). 72
Figure 3 .1e The optimisation method of the immunostaining of cld-8 TJ protein in human buttock epidermis (Magnification 20x). 73
Figure 3 .1f The optimisation method of the immunostaining of cld-12 TJ protein in human buttock epidermis (Magnification 20x). 74
Figure 3 .1g The optimisation method of the immunostaining of occludin TJ protein in human buttock epidermis (Magnification 20x). 75
Figure 3 .1h The optimisation method of the immunostaining of zo-1 TJ protein in human buttock epidermis (Magnification 20x). 76
Final optimisation of the antibodies produced the staining shown in fig 3.2.
Claudins-1, -4, -5 and -12 immunoreactivity is observed uniformerly throughout the epidermisinacharacteristic‘chickenwire’or‘honeycomb’patternofstainingofboththe granular and spinous cell layers (fig 3.2). Claudin-7 is expressed predominantly in the granular layer of the epidermis. However, in some sections there also appeared to be weaker staining in the spinous layer. Claudin-8 is expressed strongly in the uppermost layer of the epidermis and the basal layer and weakly in a ‘chicken wire’ pattern throughout the granular and the spinous cell layers of the epidermis (fig 3.2).
Immunostaining of occludin and ZO-1 is localised to the granular cell layer.
However, some immunostaining of ZO-1 is also found in the uppermost spinous layer (fig
3.2).
77
Figure 3.2 Localisation of TJ proteins in human skin. Immunostaining of photoprotected areas of human skin (buttocks) showed cld-1 (diluted 1:100), -5 (diluted 1:50) and -12 (diluted 1:50) areexpressedthroughout theepidermis in a‘chickenwire’ pattern. However, cld-4 (diluted 1:50) is more highly expressed in the basal layer of the epidermis with weaker staining throughout the epidermis. Claudin-7 (diluted1:50) appeared as chicken wire pattern mainly in the stratum granulosum. Claudin-8 (diluted 1:50) is expressed mostly in the uppermost and basal layer of the epidermis and weakly as a chicken ‘wire pattern’ throughout the granular and spinous cell layers. However, occludin and ZO-1 (diluted 1:50) are both expressed only in the top granular cell layer in a chicken wire pattern. OR All antibodies were applied at a final dilution of 1:50 except cld- 1, which was applied at a final dilution of 1:100. Magnification (20x).
78
3.2.3 Comparison of tight junction protein expression in old vs. young human skin
Having optimised the staining protocols for the TJ proteins under investigation, the study comparing their expression in young vs old skin was possible. For these two cohorts of healthy Caucasian volunteers were recruited: cohort 1, aged 18-30 years (young cohort); cohort 2, aged 65-70 (aged cohort). Both cohorts contained n=8 volunteers who were subjected to a 6mm punch biopsy from photoprotected buttock skin.
Using immunofluorescence staining, TJ proteins (clds-1, -4, -5, -7, -8, -12, occludin and ZO-1) epidermal fluorescence was quantified by image analysis (ImageJ; NIH;
Bethesda, MA, USA). When statistical analyses were carried out, no significant changes in the expression of TJ proteins was observed in aged vs. young human skin (fig 3.3a, b, c, d, e, f, g and h). Moreover, no obvious changes in the localisation of TJ proteins were observed.
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Figure 3.3a Immunofluorescent staining of cld-1 TJ protein shows no significant changes between young vs. intrinsically aged human skin. Box and whisker plots demonstrate the level of immunofluorescent staining for epidermal cld-1 in young vs. intrinsically aged, photoprotected human skin. The box identifies the inter-quartile range (25th and 75th percentiles) plus the the median (internal line). The 10th and 90th percentiles are shown as lines above and below the box, respectively. (Student’s T-test, P= 0.12; n=8). Magnification (40x).
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Figure 3.3b Immunofluorescent staining of cld-4 TJ protein shows no significant changes between young vs. aged human skin. Box and whisker plots demonstrating immunofluorescent staining of cld-4 TJ protein in young vs. aged human skin groups. The box shows the group median as a line across the middle and the quartiles (25th and 75th percentiles) as its ends. The 10th (minimum value) and 90th (maximum value) percentiles are shown as lines above and below the box, respectively. The width of the plots shows the spread of data (Student t-test, P= 0.276; n=8): Magnification (20x).
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Figure 3.3c Immunofluorescent staining of cld-5 TJ protein shows no significant changes between young vs. aged human skin. Box and whisker plots demonstrating immunofluorescent staining of cld-5 TJ protein in young vs. aged human skin groups. The box shows the group median as a line across the middle and the quartiles (25th and 75th percentiles) as its ends. The 10th (minimum value) and 90th (maximum value) percentiles are shown as lines above and below the box, respectively. The width of the plots shows the spread of data (Student t-test, P= 0.602; n=8): Magnification (40x).
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Figure 3.3d Immunofluorescent staining of cld-7 TJ protein shows no significant changes between young vs. aged human skin. Red arrow indicates stratum corneum and the white arrow indicates the stratum granulosum. The yellow arrows show a line measurement of the immunofluorescent staining of cld-7 antibody from the stratum corneum to the stratum basale (Student’sT-test, P= 0.12; n = 8). Magnification (20x).
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Figure 3.3e Immunofluorescent staining of cld-8 TJ protein shows no significant changes between young vs. aged human skin. Red arrow indicates stratum corneum and white arrow indicates stratum granulosum layer. Claudin-8 is expressed mostly in the uppermost and basal layer of the epidermis and weakly as a chicken ‘wire pattern’ throughout the granular and spinous cell layers. Therefore, a line was drawn from the stratum corneum to certain point of the stratum spinosum then another line was drawn from the stratum basale to certain point in the stratum spinosum. (Student’sT-test, P=0.85; n=8). Magnification (20x).
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Figure 3.3f Immunofluorescent staining of cld-12 TJ protein shows no significant changes between young vs. aged human skin. Box and whisker plots demonstrating immunofluorescent staining of cld-12 TJ protein in young vs. aged human skin groups. The box shows the group median as a line across the middle and the quartiles (25th and 75th percentiles) as its ends. The 10th (minimum value) and 90th (maximum value) percentiles are shown as lines above and below the box, respectively. The width of the plots shows the spread of data (Student t-test, P= 0.75; n=8): Magnification (20x).
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Figure 3.3g Immunofluorescent staining of occludin TJ protein shows no significant changes between young vs. aged human skin. (Student t-test, P= 0.14; n=8): Magnification (20x).
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Figure 3.3h Immunofluorescent staining of zo-1 TJ protein shows no significant changes between young vs. aged human skin. (Student t-test, P= 0.1; n=8): Magnification (20x).
3.2.4 A slight reduction of epidermal thickness in aged vs young human skin
Epidermal thickness has been observed in some studies to decrease with age; therefore, measures of the thickness of the epidermis in the young and aged cohorts were taken as a potential means of validating the cohort. Epidermal thickness was quantified using ImageJ Analysis software. For each section, line measurements were taken from at least five different areas from one section. Epidermal thickness was measured from the stratum basale to stratum granulosum, and excluded the stratum corneum and hair follicles
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in aged vs. young human skin. No significant difference was observed in the epidermal thickness of aged as compared to young human skin (mean ± SE; aged, 92.48µm±14.75; young, 98.13 µm±13.89;P=0.4,Student’sunpairedT-test; fig 3.4).
Figure 3.4 Epidermal thickness comparison between young and aged human epidermis. Box and whisker plots demonstrating a non significant change in epidermal thickness in young vs. aged human skin groups. The box shows the group median as a line across the middle and the quartiles (25th and 75th percentiles) as its ends. The 10th (minimum value) and 90th (maximum value) percentiles are shown as lines above and below the box, respectively. The width of the plots shows the spread of data (n=8).
3.3 Discussion
Using immunofluorescent staining, seven TJ proteins clds-1, -4, -5, -7, -8 and -12, occludin and ZO-1 - have been identified and localised in human skin (fig 3.2). This is consistent with the results from the Affymetrix® study indicating mRNA for all these
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proteins in human skin. Claudin-1 showed strong staining throughout the epidermis of human skin tissue. This observation is consistent with other studies demonstrating the existence of cld-1proteinina‘chickenwire’patternwhichischaracteristicofTJproteins.
For example, cld-1 has been demonstrated in all epidermal layers in mice (Tunggal et al.,
2005). Other studies in human tissue also demonstrate staining of cld-1 throughout the epidermis (Peltonen et al., 2007).
Claudin-4 is expressed weakly as a ‘chicken wire’ pattern throughout the epidermis. These results are broadly consistent with previous studies on the existence of cld-4 in humans and mice. As a study demonstrated that cld-4 is expressed in the granular layer of the epidermis of wild-type mice (Furuse et al., 2002), reverse transcriptase- polymerase chain reaction (RT-PCR) techniques showed the expression of cld-4 in both human skin and cultured keratinocytes (Brandner et al., 2002). Claudin-4 expression was also observed in another study, mainly in the granular layer of human skin with lower levels of expression in the spinous layer of human skin (Peltonen et al., 2007).
Although cld-5 is generally thought to be specifically expressed in endothelial cells, the Affymetrix® study also suggested that it is present in human skin. A recent study also showed the presence of cld-5 in an epithelial cell line derived from human colon (HT-
29/B6; Amasheh et al., 2005) further suggesting that this cld is not endothelial cell specific. In good agreement with the Affymetrix® study, cld-5 staining was observed in the epithelialcellsofhumanskininapartial‘chickenwire’patternthroughouttheepidermis in agreement with another study showing expression of this protein in the granular cell layers of human skin (Peltonen et al., 2007).
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In some studies, cld-7 appeared to be expressed at low level in the bottom layers of the stratum corneum (Stumpff et al., 2011), whereas other studies showed cld-7 to be expressed in all layers of the epidermis with stronger staining in the upper layers
(Kirschner et al., 2009). However, our data showed cld-7to be expressedas a ‘chicken wire’patternmostlyinthestratum granulosum.
This study is the first to demonstrate cld-8 protein expression in human skin.
Claudin-8 is seen throughout in the granular and spinous layers of the epidermis.
Interestingly, although the Affymetrix® study of Lener et al (2006) suggested that the transcript increases with age, the data described here suggest that the protein levels of cld-8 do not appear to change with age. It is usually known that changes in mRNA levels reflect changes in protein expression. However, the inconsistency between mRNA and protein expression can be caused by posttranscriptional regulation, as well as changes in mRNA and protein turnover rates (Fu et al., 2007).
This study demonstrated the expression of occludin and ZO-1 in the granular cell layer. However, some immunostaining of ZO-1 is also found in the uppermost spinous layer consistent with previous studies (Brandner et al., 2002). Previous studies in rodent skin also demonstrated that occludin and ZO-1 are found in stratum granulosum (Yoshida et al., 2001; Morita et al., 1998). Also a previous study that identified ZO-1 (by immunofluorescence) as punctuate spots confined to the uppermost cells of a rabbit corneal epithelium (Sugrue and Zieske, 1997) and normal human skin (Pummi et al.,
2001).
This study is the first to show the expression of cld-12 in human skin. Claudin-12 appearedtobeexpressedas‘chickenwire’ pattern throughout the epidermis.
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In many cases, TJ protein expression was observed throughout the epidermis.
However, electron microscopy studies demonstrate that TJs are located to the granular layer in skin. Therefore, the proteins are not all specifically located in the TJ complexes.
As mentioned in section 1.3.1 there is a precedent for this in other tissues. Claudin-4 is not specifically junctional in intestinal tissue and in the rat epididymis; neither is cld-1 restricted to the TJ (Gregory et al., 2001). This might suggest that clds may be able to influence movement of ions or solute along the lateral intercellular space without formation of TJ (Rahner et al., 2001). Alternatively, clds may have other functions that have not yet been identified. For example they could play a role in cell growth, proliferation, migration and apoptosis (Aijaz et al., 2006).
Quantification of TJ protein expression in young vs. old skin demonstrated that there is no significant change in protein expression in ageing. This is surprising given that
TJ protein expression is known to decrease in rodent tissues with increasing age.
Furthermore, skin barrier function is thought to decrease with age (Rabe et al., 2006).
Studies have shown that although TEWL measurements are generally slightly decreased in aged vs. younger individuals (Ghadially et al., 1995) the permeability of aged skin is greater than that of young skin for water soluble molecules (Tagami et al., 1972), suggesting a decrease in barrier function in ageing. However, in many of these studies, volar forearm skin was used to make the measurements so the impact of UV radiation from the sun on the permeability of skin cannot be ruled out. The fact that TJ protein expression does not change might mean that TJs are not important in the age-related changes in barrier function. Alternatively, it could be that TJ function does change with age, but this is not due to changes in protein expression. Various signalling pathways are known to regulate
TJ permeability. Age-related changes in these could putatively lead to changes in barrier 91
function without a change in the expression of TJ proteins. Unfortunately, in this study it was not possible to measure TJ function in human skin. A third, but less likely explanation is the size of the cohort. An n=8 may be too small to detect changes in expression if the changes are small. However, although possible, this explanation seems unlikely given that this n number has been found to be sufficient to detect age related changes in other proteins (Langton et al., 2010). Additionally, a cohort size of 8 was sufficient to detect changes in TJ protein expression in photoageing (chapter 4).
The epidermal thickness has been measured in young and old human skin in a number of studies. Our study showed a slight but not significant decrease in the epidermal thickness in aged compared to young human skin. A previous study also showed no significant changes in epidermal thickness comparing biopsies from the dorsal forearm, shoulders and buttocks of 71 subjects aged 20-68 years old (Branchet et al., 1990).
However, a confocal microscopy study of 34 women aged 18–69 found a significant reduction of the epidermal thickness on the back of the arm with increased age (Sandby-
Møller et al., 2003). These differences between the results are difficult to explain but could be caused due differences in the anatomical sites used for comparisons in the aged group.
In summary, several TJ proteins have been identified with staining patterns in good agreement with the literature. However, when comparing the expression and localisation of these proteins in young vs aged skin, no differences are apparent.
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4. AN INVISTIGATION INTO THE EXPRESSION OF TIGHT
JUNCTION PROTEINS IN PHOTOAGED VS.
PHOTOPROTECTED HUMAN SKIN
4.1 Introduction
The work outlined in chapter 3 demonstrated that TJ protein expression does not change in intrinsic ageing. However, skin is subject not only to normal chronological ageing, but also to extrinsic ageing. This second form of ageing is due to environmental factors such as smoking. However, the biggest effect on skin is likely to be due to UVR from sunlight. UVR can affect the skin by direct or indirect mechanisms. Direct damage can be caused to DNA (through production of dimers) and proteins (through interaction with aromatic amino acids) (Svobodova et al., 2006) and UVR also can cause indirect damage to the skin by generating ROS which cause oxidative damage to cellular macromolecules such as DNA, proteins, fatty acids and saccharides (Svobodova et al.,
2006). Skin ageing is accelerated by chronic exposure to solar UV irradiation causing photoageing. Many of the changes observed are associated with a reduced barrier function
(Helfrich et al., 2008; Holleran at al., 1997). UVR has been demonstrated to disturb the epidermal barrier both in mouse models and in man (Haratake et al., 1997; Jiang et al.,
2007; Lim et al., 2007). However, in general, the role of TJ in the UVR induced changes was not studied. More recently, one study has demonstrated that TJ function and expression is altered by exposure to acute UVB in a mouse model and in human epidermal equivalents (Yuki et al., 2011). However, to date, no study on the effects of UVR on TJ proteins has been conducted in humans.
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Therefore the aim of this study was to investigate the expression of TJ proteins in photoprotected vs. photoexposed human skin.
4.2 Results
4.2.1 Claudin-1 expression is significantly decreased in photoaged skin
To determine to extent of photoageing, clinical assessments of the skin of subjects were performed by a trained research nurse and a score was given to reflect the overall extent of photoageing. This score is based on a photonumeric scale derived by Griffiths et al, 1992 and assesses photoageing according to the following factors: fine lines and wrinkles plus dyspigmentation. The scale runs from 0 (no photoageing) to 8 (severe photoageing). Subjects with grade 6 and above were recruited for this study and considered as photoaged.
A cohort of 8 healthy, but photoaged Caucasian volunteers, age range: 65-75 years, were recruited to the study as described in section 2.2.2: Six (6) mm punch biopsies were taken from 3 distinct anatomical sites: buttock, upper inner arm and forearm. Upper inner arm is an anatomical control site to ensure that any differences found between the forearm
(photoexposed area) and hip (photoprotected area) were due to photoexposure and not to anatomical site variation. The biopsies were subsequently embedded and cryosectioned and the expression of TJ proteins were investigated using immunostaining. The specific proteins investigated were clds-1, -4, -5, -7, -8, -12, occludin and ZO-1.
Claudin-1 was highly expressed in all layers of the epidermis in a characteristic
‘chickenwire’patternofstaininginphotoprotectedbuttock sections (fig 4.1). This pattern
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of staining was maintained in upper inner arm. However, in photoaged sites, the characteristic‘chickenwire’patternofstainingwaslost.
Quantification of the total staining demonstrated a 1.7-fold reduction in total cld-1 staining in photoaged vs. photoprotected sites (ANOVA, P= 0.005, fig 4.1d).
Furthermore, comparison of signal intensity as a function of distance from the stratum corneum demonstrated that the loss of staining appeared to be greater in the basal layer of the epidermis (fig 4.2).
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Figure 4 .1 Claudin-1 expression is decreased in photoexposed vs photoprotected skin. Immunostaining demonstrated staining in the granular and spinous cells layers of the epidermis in: (a) photoprotected buttock; (b) photoprotected upper inner arm. Significantly decreased fluorescence is observed in (c) photoaged forearm compared to photoprotected buttock skin. (d) Box and whisker plots demonstrating immunofluorescent staining of cld- 1 TJ protein in human skin sections. The box shows the group median as a line across the middle and the quartiles (25th and 75th percentiles) as its ends. The 10th (minimum value) and 90th (maximum value) percentiles are shown as lines above and below the box, respectively. The width of the plots shows the spread of data. Quantification reveals a 1.7 fold reduction in cld-1 expression in photoaged skin vs photoprotected skin (ANOVA, P=0.005; n=8): Magnification (40x).
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Figure 4 .2 A reduction in the expression of cld-1 in the basal layer in photoaged skin. Line measurments from stratum corneum showed a siginificant reduction in the basal layer of photoaged skin compared to photoprotected area of human skin (ANOVA, P= 0.00025; n=8) using ImageJ analysis.
4.2.2 Claudin-7 is significantly increased in photoaged skin
In photoprotected skin, cld-7 is expressed predominantly in the granular layer of the epidermis with some staining in the spinous layer. Interestingly, in upper inner arm (the anatomical site control) the intensity of the staining was significantly higher compared to that observed in buttock skin (fig 4.3a, 4.3b). In photoaged forearm, the intensity of the staining was significantly increased by a further 1.5 fold compared to upper inner arm
(ANOVA, P= 0.019; fig 4.3c, 4.3d). Moreover, the expression of cld-7 was observed also to expand to the spinous layer of the epidermis in photoaged skin.
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Figure 4 .3 Claudin-7 is increased in photoaged skin. Immunostaining reveals granular layer staining in: (a) photoprotected buttock and (b) photoprotected upper inner arm. Increased intensity of staining was observed in (c) photoaged forearm 1.5 fold higher compared to photoprotected section UIA. (d) Quantification reveals that this increase is significant (ANOVA, P=0.019; n=8): Magnification (20x).
4.2.3 Claudin-12 is significantly increased in photoaged skin
Claudin-12, like cld-1, is expressed in both the granular and spinous layers of the epidermis in photoprotected skin (fig 4.4a, 4.4b). In photoaged forearm, the intensity of the staining was significantly higher by 1.6 fold compared to that seen in photoprotected buttock skin (ANOVA, P= 0.004; forearm fig 4.4c, 4.4d). However, significantly lower expression of cld-12 was in the upper inner arm observed compared to the buttocks. That
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may indicate anatomical differences in cld-12 expression. Also, no obvious changes in the localisation of cld-12 were observed.
Figure 4 .4 Intensity of cld-12 staining is significantly increased in photoaged skin. Immunofluorescence demonstrates staining in the granular and spinous cells layers of the epidermis: (a) photoprotected hip; (b) photoprotected upper inner arm. 1.6 fold increased fluorescence is observed in (c) photoaged forearm compared to buttock. (d) Box and whisker plots demonstrating immunofluorescent staining of cld-12 TJ protein in human skin sections. The box shows the group median as a line across the middle and the quartiles (25th and 75th percentiles) as its ends. The 10th (minimum value) and 90th (maximum value) percentiles are shown as lines above and below the box, respectively. The width of the plots shows the spread of data. Quantification reveals that this increase is significant (ANOVA, P=0.004; n=8): Magnification (20x). However, in the anatomical site control, cld-12 expression is reduced compared to that observed in buttock skin.
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4.2.4 No significant changes are observed in other tight junction proteins in
photoaged skin
Using immunofluorescent staining, expression of other TJ proteins (clds-4, -5, -7, -
8, occludin and ZO-1) were quantified in photoaged and photoprotected human skin (data not shown). However, no significant changes were observed in their levels of expression or pattern of localisation.
4.2.5 A reduction in claudin-1 and an increase in claudin-12 mRNA levels is
observed in photoaged human skin
Since changes in the expression of clds-1, -7, and -12 proteins were observed in photoaged human skin, qPCR was carried out to investigate whether the gene expression level of these particular proteins varied in photoaged and photoprotected human skin
(n=6).
As shown in fig 4.5, expression of the (cld-1) gene was significantly lower in photoaged skin compared to photoprotected buttock skin (fig 4.5). However, a significant reduction in cld-1 was also noticed in the upper inner arm compared to the buttocks that may indicate anatomical difference in the expression of cld-1. Moreover, cld-12 gene expression was higher in photoaged compared to photoprotected skin. These results are consistent with the changes in the expression levels of these proteins in photoaged skin.
However, no obvious change in cld-7 gene expression was observed in photoaged compared to photoprotected human skin.
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Figure 4 .5 Quantitative PCR analysis of TJ proteins clds-1 and -12 showed changes in their expression level in photoaged human skin. The fold-difference indicates a reduction in cld-1 and an increase in cld-12 gene expression in photoaged human skin. However, no significant difference was observed in cld-7 in photoaged and photoprotected skin.
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4.3 Discussion
4.3.1 Expression and localisation of claudins-1, -7 and -12 in photoprotected and
photoaged skin
This study, the first in humans, investigated the effects of photoageing on TJ protein expression in the epidermis and demonstrates significant differences in cld expression between photoaged and photoprotected human skin i.e. higher expression of clds-7 and -12 and lower cld-1 expression in photoaged forearm skin.
The reduction in cld-1 might suggest that a disruption of the TJ barrier occurs as a result of UVR exposure considering the important role of cld-1 as a barrier illustrated by the cld-1 knock out mouse (Furuse et al., 2002). Additionally, loss of cld-1 is observed in the human conditions atopic dermatitis and the human genetic equivalent of the cld-1 knock out mouse, Neonatal Icthyosis Sclerosing Cholangitis syndrome (NISCH, Hadj-
Rabia et al., 2004). Both these conditions are associated with poor epidermal barrier function. Furthermore, in photoaged skin, a significant increase is also observed in the expression of two other clds-7 and -12.
Claudin-7 is considered as being a pore-forming isoform because it’s over expression has been shown to increase ionic permeability through the TJ (Brandner, 2009).
Moreover, overexpression of cld-7 in cultured LLC-PK1 (a renal epithelial cell line originally derived from porcine kidneys) has been shown to increase paracellular Na+ conductance through TJs (Alexandre et al., 2005).
A recent study also demonstrated a specific functional role for cld-12. In the enterocyte cell line, CaCo-2, cld-12 expression is induced by vitamin D. This increase in cld-12 enhanced transepithelial Ca2 transport (Fujita et al., 2008). Interestingly, the
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epidermis is the major site for Vitamin D synthesis promoted by sun light exposure
(Holick et al., 1980); therefore it is tempting to speculate that this may explain the overexpression of cld-12 observed in photoaged skin. In vitro studies may be useful in uncovering whether cld-12 is vitamin D inducible in skin as well as the gut.
Taken together, the data suggest that photoageing is associated with loss of cld-1, an important structural component of TJs and increases in the expression of two pore forming clds. Overall, these data might suggest that these changes in the repertoire of expressed TJ components in photoaged human skin may have functional consequences for the overall integrity of the epidermal barrier. However measuring the barrier function of the skin will provide a better understanding of the effects of these changes on the epidermal barrier function.
The decreases in cld-1 and the increase in cld-12 gene expression in photoageing showed using qPCR are consistent with the changes to protein expression levels observed using immunofluorescent staining of photoaged and photoprotected human skin. This suggests that at least part of the mechanism by which cld protein levels change is due to transcriptional changes. However, no obvious change in cld-7 message levels is found in photoaged skin. This may be due to the low expression levels of cld-7 mRNA in the epidermis as observed in the Affymetrix studies in section 3.2.1. However, due to lack of sufficient mRNA, qPCR could not be repeated at higher mRNA concentrations.
Alternatively, it is possible that cld-7 gene expression may not change as a result of UVR and the change in protein levels is due to post translational events. There is a precedent for this in a study looking at claudin levels in tumours, breast tumours showed no changes in cld-4 mRNA expression compared with normal breast tissue but the protein expression of cld-4 was reduced in tumour cells (Tokés et al., 2005). However, the underlying 103
mechanism was not explored. This shows that claudin protein can be regulated independently of gene expression.
UVR has been demonstrated in many studies to affect both gene and protein expression. For example, a study showed that UVR increases the expression of Mi-2
ATPase (the core subunit of the nucleosome remodeling deacetylase (NuRD) complex) in the skin by a direct effect on its gene expression following UVR exposure (Burd et al.,
2008). Another study has also showed upreglation of IL-15 following UVB exposure as a result of an increase in mRNA level of IL-15 (Mohamadzadeh et al., 1995). Moreover, the induction of snail and slug expression is caused by an increase in their mRNA levels following UVR exposure. This induction was suggested to be partly by p38 MAPK cascades (Hudson et al., 2007). Hence UVR is known to regulate gene expression leading to changes in protein expression levels. However, UVR can also directly affect protein levels independently of transcriptional changes. For example, downregulation of aquaporin-3 (AQP3) protein occurs as a result of exposure of keratinocytes to UVR. This is accompanied by changes to the water permeability of the keratinocytes (Cao et al.,
2007). This loss of AQP3 is due to reactive oxygen mediated actions on signaling pathways leading to loss of AQP3 in keratinocytes. Another mechanism by which UVR induces changes to protein levels is via induction of matrix metalloproteinase activity.
These enzymes are found in the epidermis and the dermis and degrade extracellular matrix proteins such as collagen (Fisher et al., 1997). Thus, collagen levels decrease due to the action of these enzymes but the message levels remain stable.
Work in the Dept of Dermatology in Manchester has also demonstrated that UVR can directly damage proteins such as fibronectin due to the high content of potential chromophores within the protein (Sherratt et al,. 2010). 104
Thus UVR can induce changes to protein levels either through modulation of gene expression levels or via direct effects on protein levels. Hence, either or both mechanisms may explain the changes to claudin levels noted in photoaged vs photoprotected skin.
Further work will be required to understand the relative contributions of transcriptional vs post translational effects on claudin expression levels when skin is exposed to UVR.
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5. CHARACTERISATION OF TIGHT JUNCTION PROTEIN
EXPRESSION IN HUMAN SKIN SUBJECTED TO A SINGLE,
ACUTE DOSE OF UVB
5.1 Introduction
The data presented in chapters 3 and 4 demonstrate that TJ protein expression is not changed in chronological ageing but is aberrant in extrinsically aged (photoaged) skin compared to photoprotected skin. Barrier function in skin is known to be altered in response to acute doses of UVB. For example, a study in hairless mice demonstrated a time- and dose-dependent increase in TEWL when the mice were irradiated with single doses of UVB. This suggests a disruption in the barrier function of the skin in response to
UVB (Haratake et al., 1997). Other studies - in human skin equivalents and keratinocytes cultures - have also demonstrated changes to TJ protein expression in response to single doses of UVB (Yuki et al., 2011). However, the effects of acute irradiation on TJ protein expression have not previously been studied in human tissue.
Although UVR from sunlight is undoubtedly the biggest environmental stimulus to skin, other factors such as smoking, pollution, repetitive facial movements etc may also have an impact. Additionally, chronic effects may be different to acute ones. Therefore, in this chapter, the impact of a single dose of UVB on TJ protein expression in human volunteers was investigated. This study was performed both to confirm that UVR impacts upon TJ proteins in skin but also to compare acute versus chronic effects.
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5.2 Results
5.2.1 The expression of claudin-1 is significantly reduced in response to a single dose
of UVB
As outlined in section 2.2.3, healthy human volunteers were exposed to a single dose of 200mJ/cm2 (approximately 3MED) of UVB applied to photoprotected buttock skin. Biopsies were then taken at 4- and 24-hours post-irradiation along with a single biopsy from the opposing, contralateral buttock which had not been irradiated. The biopsies were cryosectioned and stained using immunofluorescence for clds-1, -7 and -12, as these proteins were shown to be altered in response to chronic UVR i.e. in photoaged skin.
The image and quantification in fig 5.1a-d demonstrates that at 4-hours, no detectable change was observed in cld-1 levels or localisation. However, at 24-hours, a significant reduction in cld-1 levels was observed (ANOVA; P=0.001).
Analysis of the images appeared to show that most of the UVB-induced loss of cld-
1 occurred in the basal layer of the epidermis. Therefore, to further investigate this, plots of signal intensity versus distance from the stratum corneum confirmed that this loss of cld-1 was most pronounced in the basal layer of the epidermis (fig 5.2).
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Figure 5.1 Claudin-1 expression is decreased following acute UVB irradiation of human skin. Photoprotected buttock skin was irradiated with 200mJ/cm2 UVB and biopsies taken at 4- and 24-hours post-UVB. At 4-hours (b) there was no significant difference in cld-1 expression or localisation compared to unirradiated skin (a). However, at 24-hours (c) there was a 1.6x decrease in staining intensity. Box and whisker plots demonstrating immunofluorescent staining of cld-1 TJ protein in acutely irradiated human skin (d). The box shows the group median as a line across the middle and the quartiles (25th and 75th percentiles) as its ends. The 10th (minimum value) and 90th (maximum value) percentiles are shown as lines above and below the box, respectively. The width of the plots shows the spread of data (AVOVA; P=0.001; n=8): Magnification (40x).
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Figure 5.2 Claudin-1 expression is significantly decreased in the basal layer following acute UVB irradiation. Mean intensity of cld-1 showed significant reduction particularly in the basal layer of UVB exposed skin at 24-hours post-irradiation (ANOVA, P=0.0003; n=8). However, no significant change in cld-1 expression was observed at 4-hours post- irradiation.
5.2.2 The expression of claudins-7 and -12 are unaltered in response to a single dose
of UVB
Claudin-7 expression at 4-hours post-irradiation showed no change in the level or localisation of the protein. At 24-hours post-UVB, there was a small, but insignificant increase in cld-7 staining (fig 5.3 a-d).
Similarly, no change was observed in the expression or localisation of cld-12 at 4- or 24-hours post-UVB irradiation (fig 5.4 a-d).
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Figure 5.3 UVB irradiation does not significantly alter cld-7 expression. Photoprotected buttock skin was irradiated with a single dose of 200mJ/cm2 UVB and biopsies taken at 4-hours (b) or 24-hours (c) post-irradiation. Claudin-7 staining in these biopsies was compared with that observed in unirradiated skin from the opposing buttock (a). Quantification (d) of the images identified no difference in the staining for this TJ protein: Magnification (40x).
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Figure 5.4 UVB irradiation does not significantly alter cld-12 expression. Photoprotected buttock skin was irradiated with a single dose of 200mJ/cm2 UVB and biopsies taken at 4-hours (b) or 24-hours (c) post-irradiation. Claudin-12 staining in these biopsies was compared with that observed in unirradiated skin from the opposing buttock (a). Box and whisker plots demonstrating immunofluorescent staining of cld-12 TJ protein in acutely irradiated human skin (d). The box shows the group median as a line across the middle and the quartiles (25th and 75th percentiles) as its ends. The 10th (minimum value) and 90th (maximum value) percentiles are shown as lines above and below the box, respectively. The width of the plots shows the spread of data. Quantification of the images showed a small increase in cld-12 expression at 24-hours, but this was not significant (ANOVA, P= 0.095; n=8): Magnification (40x).
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5.3 Discussion
To my knowledge, this is the first study in humans investigating the effects of an acute dose of UVB on TJ protein expression in skin and demonstrates changes in claudin levels. These changes are unlikely to be due to excessive burning of the skin because the dose given was only 3 MED (see table 5.1).
(200mJ/cm²) this dose induced clinical effects including erythema and induced migration of Langerhans cells out of the epidermis after 24 hours from UVB exposure in
PLE (Blackburn et al., 2005). Moreover, significant induction of Caspase-3 (a marker of apoptosis) expression observed after 24 hours of acutely irradiated human skin (Tye et al.,
2006). Therefore, a dose of 200 mJ/cm² had a biological effect but can be encountered under normal sunlight exposure.
Table 5 .1 The MED values for acutely irradiated human skin.
Subject MED MED/(200mJ/ The mean of MED for Voulnteers Age Initial (mJ/cm²) cm²) 200 mJ/cm² Patient 1 PH2 51 42 4.761904762 3.375527426 Patient 2 RB 43 42 4.761904762
Patient 3 IS 43 60 3.333333333
Patient 4 SO 35 70 2.857142857
Patient 5 WS 33 84 2.380952381
Patient 6 LF 40 70 2.857142857
Patient 7 RS 45 36 5.555555556
Patient 8 PH 33 70 2.857142857
Mean for MED 59.25
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The results demonstrate that even at 24-hours post-irradiation, human skin suffers significant loss of cld-1. Furthermore, most of this loss occurs in the basal layer of the epidermis. This is in good agreement with observations made in photoaged human skin where cld-1 was also observed to be reduced in photoaged versus photoprotected human skin (fig 4.1).
Why loss of cld-1 occurs most significantly in the basal layer is at present unclear.
It is possible that cells in the basal layer, being more proliferative, may simply be more susceptible than cells higher up the epidermis that are by definition, more differentiated.
However, there are examples in the literature to suggest that the expression of other UVR responsive proteins with a transepidermal expression pattern are not affected by UVR anymore significantly in the basal layer cells than in suprabasal layers. For example, heparanase (a unique mammalian enzyme known to cleave heparan sulfate (HS), the principle polysaccharide at the cell surface and ECM of different types of tissues (Iozzo et al., 2001)), like cld-1 is expressed throughout the epidermis and is UVB responsive.
However, the expression of heparanase is up-regulated in response to UVB equally in all layers of the epidermis (Kurdykowski et al., 2012). This might suggest therefore, that there is a specific mechanism causing greater changes in basal cld-1 levels than is observed in the rest of the epidermis in response to UVB. A study showed that p63, a gene regulatory factor, is mostly expressed in the basal layer of the epidermis and is down regulated in response to UVB irradiation (Liefer, et al., 2000). Moreover, a recent study showed a reduction in cld-1 expression in p63 deficient keratinocytes along with a disruption to the barrier function (Lopardo et al., 2008). In another study, cld-1 was shown to be a target of matrix metalloproteinase-9 (MMP-9) which is also expressed mainly in the basal layer of the epidermis (Roh et al., 2012). Hence some of these observations could be possible
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mechanisms of the reduction seen in cld-1 in the basal layer of the epidermis upon UV irradiation.
Interestingly, changes in clds-7 and -12 that have been shown to increase in photoaged skin were not significant in response to acute UVB irradiation. This potentially suggests that the mechanisms involved in altering specific cld isoforms may be different.
However, although the changes were not significant, there was a trend towards an increase in the expression of both clds-7 and -12. Hence it is possible that biopsies taken at time points greater than 24-hours post-irradiation may yield significant changes in these two clds.
In CaCo-2 cells, vitamin D is known to induce expression of cld-12 and this enhances transepithelial Ca2 transport (Fujita et al., 2008). This may explain the over expression of cld-12 in photoaged skin since the epidermis is the major site for vitamin D synthesis promoted by sun light exposure (Holick et al., 1980). However, to date, the relationship between vitamin D and cld-12 expression has not been investigated so it may be a gut-specific phenomenon.
Overall, the pattern of changes to cld expression is consistent with a possible decrease in the barrier function of skin. Claudin-1 is important in the permeability barrier of skin to water; clds-7 and -12 are known to be pore forming clds in other tissues
(Brandner, 2009). Taken together, this suggests that the changes in response to acute UVB may be consistent with reduced barrier function and may partly explain the known changes to skin barrier function observed in response to acute UVB in mouse models and in one human study (Haratake et al., 1997, Lim et al., 2007).
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In conclusion, the changes observed in chronically photoexposed skin also occur in response to acute UVB doses. This may suggest that acute UVB sets in motion changes that are difficult to fully reverse or repair, thus explaining the alterations in cld expression observed in chronic photoageing.
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6. ULTRAVIOLET-B IRRADIATION MODULATES TIGHT
JUNCTION PROTEIN EXPRESSION IN CULTURED PRIMARY
KERATINOCYTES
6.1 Introduction
The data in chapter 4 show significant changes in the expression of TJ proteins in photoaged versus photoprotected human skin. Additionally, the data in chapter 5 demonstrates that acute exposure of skin to ultraviolet radiation (UVR) also induces very similar changes to TJ protein expression as observed in photoageing. Interestingly, the loss of cld-1 in skin acutely exposed to UVB was most pronounced in the basal layer of the epidermis perhaps suggesting that keratinocytes are more or less susceptible to UVR depending upon their state of differentiation.
The overall changes to TJ protein expression in skin in response to UVR may suggest an alteration (possibly a reduction) in the TJ barrier function of skin. However, TJ function in human skin is difficult to measure. Additionally, ethical constraints in terms of access to human tissue make modelling changes in TJ protein expression with respect to time challenging.
Therefore, in this chapter, a model of skin has been utilised. This model is normal human epidermal primary keratinocytes (NHEK) maintained in culture. These cells were exposed to doses of UVR (primarily UVB; Philips TL-12), to assess the effects on TJ protein expression, TJ function and recovery post-UVR. Additionally, the responses of differentiated versus undifferentiated NHEK to UVR were investigated.
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6.2 Results
6.2.1 UVB irradiation of undifferentiated NHEK alters the expression of tight
junction proteins
The effects of UVR on the viability of NHEK were examined in the first instance.
This experiment was conducted to identify sub lethal doses of UVB because it was important to establish that any effects observed in response to irradiation were due to effects on TJ proteins specifically, and not due to cell death.
Primary keratinocytes were grown to 90% confluency. Following this they were irradiated with a range of UVB doses (0-20 mJ/cm²; Philips TL-12; 270-400nm). Twenty- four hours post-irradiation, the viability of the cells was established using an MTT assay as described in section 2.7.1. The data in fig 6.1 demonstrate that doses of more than 15 mJ/cm² significantly reduced cell viability (ANOVA, P=0.001; n=3). However, at lower doses, the viability of NHEK was not significantly different to that of control, unirradiated cells (fig 6.1).
Having established that doses of 15mJ/cm2 or lower have no effect on cell viability, in the next experiment, the effects of UVB on TJ protein expression were established using
Western blotting. The blots were subsequently analysed using densitometry so that any changes observed could be quantified. Primary keratinocytes express a limited number of
TJ proteins compared to human skin. Therefore, the expression of clds-1, -4, occludin and
ZO-1onlywereinvestigated.Additionally,β-actin (a housekeeping protein) levels were analysed as a loading control.
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Figure 6.1 UVR at doses below 20mJ/cm2 does not affect the viability of primary keratinocytes. Primary keratinocytes were irradiated with various doses of UVB and viability measured 24-hours post-irradiation using an MTT assay. At doses of 15mJ/cm2 or lower, the viability was not significantly different to that of unirradiated cells. However, at a dose of 20mJ/cm2 cell viability was significantly lower than that of control cells (ANOVA, P=0.001; n=3).
The blot and densitometry in fig 6.2 show that at doses as low as 5mJ/cm2, cld-1 expression was significantly reduced in irradiated versus unirradiated NHEK (38% lower in irradiated versus unirradiated cells; ANOVA, P=0.02; n=4). Conversely, the expression of cld-4 was significantly increased in irradiated as compared to unirradiated cells at doses as low as 5mJ/cm2 (up to 247% higher in irradiated versus unirradiated cells; ANOVA,
P=0.006; n=4) (fig 6.2). No significant changes were found in the levels of occludin, ZO-1 andβ-actin in UV-irradiated versus unirradiated keratinocytes.
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Figure 6.2 Tight junction protein expression in response to UVR in undifferentiated keratinocytes. The expression of cld-1 was reduced in irradiated vs unirradiated cells (ANOVA, P=0.02; n=4). In contrast, cld-4 expression increased in irradiated vs. unirradiated cells (ANOVA, P=0.006; n=4). However, no changes to occludin, ZO-1 and β-Actin protein level were observed in response to irradiation.
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6.2.2 Tight junction protein expression does not recover completely over time
Having demonstrated that TJ protein expression is altered in response to UVR, in the next experiment, the time taken for expression levels to return to normal was investigated.
Cells were irradiated with 15mJ/cm2 UVR and cell lysates produced at 24-, 48-, 72- and 96-hours post-irradiation. Tight junction protein expression was then measured at these time points relative to that in unirradiated, control cells.
In irradiated cells, even at 96-hours post-irradiation, cld-1 levels were still significantly lower than in unirradiated cells (ANOVA, P=0.005; n=3, fig 6.3). Similarly, cld-4 levels were still higher in irradiated cells than unirradiated cells at 96-hours post-
UVR (ANOVA, P=0.04; n=3, fig 6.3).However,β-actin levels did not change in the cells over 96-hours post-irradiation.
6.2.3 UVR does not affect the expression of tight junction protein mRNA
Since UVR affects the expression of clds-1 and -4 within 24-hours of irradiation, the next experiment was performed to establish whether this was an effect on the expression of the transcripts, or some post transcriptional change.
The gene expression of clds-1 and -4 were investigated 24-hours post-irradiation with a dose 15mJ/cm2 using qPCR. As shown in fig 6.4a, the expression of both clds-1 and
-4 did not change in irradiated or unirradiated NHEK cells (ANOVA, n=3).
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Figure 6.3 The expression of clds-1 and -4 does not return to baseline levels within 96 hours of irradiation. The recovery post-UVR exposure of undifferentiated keratinocytes was investigated over 4 days. However, levels did not return to baseline within 96 hours of irradiation.
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Figure 6.4 Quantitative PCR analysis of TJ proteins clds-1 and -4 showed no changes in their expression in irradiated keratinocytes. The fold-difference indicates no changes in both cld-1 and cld-4 expression in irradiated (D= 15mJ/cm2) compared to unirradiated keratinocytes (ANOVA, n=3).
6.2.4 Differentiated keratinocytes showed no significant changes in tight junction
protein expression in response to UVR
The data obtained in human skin suggests that the extent of loss for cld-1 may depend on the differentiation state of keratinocytes as cells in the basal layer suffered much greater losses of cld-1 than cells in higher layers of the epidermis (fig 5.2). To test this hypothesis, the effects of UVR on differentiated keratinocytes were next investigated.
Primary keratinocytes can be induced to differentiate by raising the extracellular calcium concentration, a technique called ‘calcium switch’ (Pillai et al., 1990). Hence,
NHEK were induced to differentiate for 24-hours by increasing the calcium concentration in the medium to 1.2mM. The cells were then irradiated with UVR at varying doses and the cell viability in response to UVR was investigated using MTT assay. The graph in fig
6.5 demonstrates that doses below 20mJ/cm2 do not significantly affect cell viability
(ANOVA, P=0.0001; n=3) (fig 6.5).
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Figure 6.5 UVR at doses below 20mJ/cm2 does not significantly affect the viability of differentiated keratinocytes. Differentiated keratinocytes were irradiated with various doses of UVB and viability measured 24-hours post-irradiation using an MTT assay. At doses of 15mJ/cm2 or lower, the viability was not significantly different to that of unirradiated cells. However, at a dose of 20mJ/cm2 cell viability was significantly 80% lower than that of control cells (ANOVA, P=0.0001; n=3).
Next, the effects of UVR at different doses on TJ protein expression were investigated in NHEK that had been differentiated for 24-hours pre-UVR exposure. Protein was extracted from the cells 24-hours post-irradiation and the levels of TJ proteins investigated using western blotting and densitometry.
The data in fig 6.6 demonstrate that doses up to 20mJ/cm2 resulted in no significant change in the protein levels of clds-1, -4, occludin or ZO-1 at 24-hours post-irradiation.
Next, to see if the timing of irradiation relative to calcium differentiation was important, undifferentiated NHEK were irradiated and then immediately placed in high calcium-containing medium. When TJ protein expression was investigated in these cells,
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no change in expression levels of any TJ protein was observed at any dose of UVR (fig
6.7).
Figure 6.6 UVR does not affect expression of TJ proteins in differentiated keratinocytes. Primary keratinocytes grown in 1.2mM calcium for 24-hours prior to irradiation were irradiated with a single dose of 0, 5, 10, 15 or 20mJ/cm2. No significant change in clds-1, -4, occludin and ZO-1 expression was observed at any dose.
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Figure 6.7 Calcium switch abolishes the effects of UVR on TJ protein expression in keratinocytes. Undifferentiated keratinocytes were irradiated and then placed in medium containing high calcium. No significant changes in clds-1, -4, occludin and ZO-1 expression were demonstrated.
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6.2.5 UVR causes reductions in the barrier function of keratinocytes
The use of primary human keratinocytes offers the possibility of measuring TJ function in response to UVR because simple assays are available for use in cell monolayers. One such assay is measurement of TEER which is a very well established measure of TJ function (Guzman-Aranguez et al., 2012). The basis of the assay is that the electrical resistance of the monolayer is a proxy for TJ function. In general the TEER of the monolayer is inversely proportional to the TJ permeability. Hence, as TJs become less permeable, TEER increases. Conversely, as permeability goes up, TEER is reduced.
In NHEK, TEER increases in response to differentiation of the cells i.e. in response to calcium switch. Therefore, to measure the effect of UVR on TJ function, TEER measurements were made in irradiated versus unirradiated cells at different times post calcium switch.
Keratinocytes were cultured on TranswellTM membranes in keratinocyte basal medium. Once confluent, the cells were switched to medium containing 1.2mM calcium and TEER measurements were taken with time. At three days post-calcium switch, the
TEERofthemonolayerwasapproximately100Ω/cm2 higher than at day zero showing the development of functional TJs (fig 6.8a ‘control’)(ANOVA,P<0.01;n=6). To determine the functional effect of UVR, cells were irradiated at 24-hours post-calcium switch with a dose of 15mJ/cm2. In these cells, TEER dropped significantly for the next 24-hours but then increased again. However, TEER levels did not recover to those seen in unirradiated cells (fig 6.8b ‘irradiated’)(ANOVA,P<0.01;n=6).
Cells were next irradiated immediately before switching to high calcium. In these cells, TEER developed in a manner identical to that seen in unirradiated cells (fig 6.8c).
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Figure 6.8 UVR disrupts TJ function depending on the timing of irradiation. (a). TEER measurements increase over time from high calcium switch (ANOVA, P < 0.01; n=6). (b) TEER develops over four days post-calcium switch in keratinocytes (control) (ANOVA, P < 0.01; n=6). However, if cells are irradiated 24 h after placing them in medium containing high calcium (1.8mM) (Dose 15), TEER reduces below that of control levels. However, if cells are irradiated and then calcium switched (c), TEER develops normally.
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6.2.6 UVR induces disruptions to the localisation of tight junction proteins in
keratinocytes
Figure 6.8b shows that UVR reduces TJ barrier function depending on the timing of irradiation relative to calcium switch. However, fig 6.7 shows that in differentiated cells,
UVR has no effect on TJ protein expression levels. Therefore, the possibility that UVR may alter the localisation of TJ proteins was next investigated by performing immunofluorescent staining of TJ proteins in irradiated and unirradiated NHEK.
Differentiated keratinocytes were cultured in 8-well chamber slides and irradiated at doses of 0 and 15 mJ/cm², 24-hours post-calcium switch. Then expression of clds-1, -4, occludin and ZO-1 were observed over 3-days post-irradiation. In unirradiated NHEK, cld-
1 was localized at the cell membrane in a continuous pattern of staining in differentiated keratinocytes. Similar results were found for cld-4, occludin and ZO-1. However, nuclear staining was also found in cells stained for both occludin and ZO-1. Interestingly, in cells stained 24-hours post-irradiation with a dose of 15mJ/cm2, cld-1 was expressed in a discontinuous pattern at the cell membrane with obvious breaks in the pattern of staining
(fig 6.9a ‘arrows’). Furthermore, small pools of cld-1 appeared in the interior of the cells.
This pattern of staining did not appear to alter with time post-irradiation and the cld-1 staining in irradiated cells at two and three days post-UVR appeared similar to that in cells at 24-hours post-irradiation. No changes were found in the staining patterns for cld-4, occludin and ZO-1 in irradiated and unirradiated keratinocytes (fig 6.9b, c, d).
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Figure 6.9a The effect of UV irradiation on the organization of cld-1 TJ protein in NHEK cells. A continuous pattern of staining observed at the cell membrane in unirradiated keratinocytes. However, an alteration in the organisation of cld-1 was observed in irradiated differentiated keratinocytes (arrows). Moreover no further obvious changes were observed over 3 days from irradiation.
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Figure 6.9b The effect of UV irradiation on the organisation of cld-4 TJ protein in NHEK cells. A continuous pattern of staining is observed at the cell membrane in unirradiated keratinocytes. However, no obvious alteration in the organisation of cld-4 is observed in irradiated differentiated keratinocytes.
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Figure 6.9c The effect of UV irradiation on the oragnisation of occludin TJ protein in NHEK cells. A continuous pattern of staining observed at the cell membrane along with nuclear staining in unirradiated keratinocytes. However, no obvious alteration in the organisation of occludin observed in irradiated differentiated keratinocytes.
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Figure 6.9d The effect of UV irradiation on the organisation of ZO-1 TJ protein in NHEK cells. A continuous pattern of staining observed at the cell membrane with nuclear staining in unirradiated keratinocytes. However, no obvious alteration in the organisation of ZO-1 observed in irradiated differentiated keratinocytes.
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Next to investigate the effects of inducing differentiation at the same time as irradiation; TJ protein expression and localisation were investigated in keratinocytes that were irradiated at the same time as the calcium switch. However, no obvious changes were found in TJ protein organisation in irradiated versus unirradiated differentiated keratinocytes (data not shown).
6.3 Discussion
The human studies outlined in chapters four and five suggested that UVR might have differential effects on TJ protein expression depending on the differentiation state of keratinocytes. In this chapter, this possibility has been investigated using a primary human keratinocyte model exposed to different media conditions which induce a more differentiated or undifferentiated phenotype. In addition, functional measurement of TJs in response to UVR was also investigated.
The data described in this chapter demonstrate that undifferentiated keratinocytes undergo aberrant TJ protein expression in response to sub lethal doses of UVR.
Specifically, cld-1 expression is significantly reduced whilst cld-4 expression increases.
This is in close agreement with data obtained in human tissue where cld-1 was also observed to decrease both in responses to a single dose of UVR and in chronically photoexposed human skin. Increasing expression of cld-4 in irradiated keratinocytes is somewhat surprising because no change was observed in the expression of this protein in photoaged human skin. However, due to limitation of the availability of tissue, cld-4 was not investigated in acutely irradiated tissue. Therefore, it is possible that in acutely irradiated human skin this protein may also be upregulated but subsequently returns to
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baseline levels and is therefore not seen to be altered in chronic photoageing. Alternatively, changes in cld-4 levels may be an artefact of using cells in culture. Further work will therefore be required to understand the relevance of increasing cld-4 levels to human skin.
Keratinocytes in culture express a limited subset of clds compared with skin and clds-7 and -12 (observed to increase in photoaged skin and acutely irradiated skin) are not expressed in keratinocytes. Therefore it was not possible to investigate these proteins in the keratinocyte model. However, interestingly, cld-4, like cld-7 which is increased in skin in response to UVR, is implicated in the movement of ions through the paracellular pathway.
Therefore, it is possible that the response of keratinocytes to UVR is similar to that of skin i.e. reductions in cld-1 and upregulation of isoforms involved in ion movement. However, the exact isoform upregulated in keratinocytes is not the same as in human skin.
The mechanism by which keratinocytes undergo altered cld expression in response to UVR is not clear but is unlikely to be due to transcriptional effects. This is because
UVR did not induce changes to TJ mRNA expression. Furthermore, the levels of other TJ proteins (ZO-1 and occludin) and the housekeeping gene β-actin remained constant following a dose of UVR. Therefore, changes in protein synthesis may also largely be discounted. The possibility therefore exists that UVR has direct effects on the existing pools of protein within the cell. Decreases in cld-1 and simultaneous increases in cld-4 may suggest effects of UVR on proteins involved in regulating cld degradation within cells. Putatively, cld-4 may be stabilised thus preventing its degradation whilst cld-1 degradation is increased. Other studies have demonstrated that UVR generates reactive oxygen species leading to the activation of matrix metalloproteinases (MMPs) (Fisher et al., 1997). Putatively, cld-1 but not -4 may be a target of MMPs. At present, very little is known regarding the molecular mechanisms which regulate existing cld pools within cells 134
and further work would be required to fully understand the differential regulation of cld isoforms in response to UVR.
In contrast to undifferentiated keratinocytes, cells allowed to differentiate for 24- hours in high calcium-containing medium do not undergo changes to TJ protein expression in response to UVR. Previous studies by others have demonstrated that ‘calcium switching’ induces assembly of cellular junctions including adherence junctions and desmosomes (Pillai et al., 1990). The present study also demonstrates that calcium induces
TJ formation as evidenced by the rise in TEER on switching the cells to high calcium- containing medium. Thus the resistance of differentiated cells to changes in cld expression may be explained by assembly of the individual proteins into TJ complexes. Conceivably, clds existing outside of TJ complexes (as would be the case in undifferentiated cells) may be vulnerable to the cellular machinery affecting their levels. However, once assembled intoTJcomplexes,theyareineffect‘protected’fromwhatevertheagentsarethatmodify expression levels in undifferentiated cells.
Whatever the mechanisms involved, it is clear from these studies that the effects of
UVR on TJ protein expression are more severe in undifferentiated versus differentiated keratinocytes. This is in close agreement with the observation in human skin in which most loss of cld-1 occurs in the basal layer keratinocytes (fig 5.2).
The functional effects of UVR on TJs depend on the timing of irradiation relative to calcium switch. In cells committed to production of TJs i.e. calcium switched for 24- hours before irradiation, UVR induces a pronounced decrease in the TJ barrier function as evidenced by the reduction in TEER. The loss of barrier function may be due to disruption of the cld staining pattern. Thus, although the absolute levels of cld-1 are similar as judged
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by Western blotting, much of the cld-1 is mislocalised as a result of irradiation and not available to make functional TJs. Such disruptions to cld localisation have also been observed by others. A recent study by Yuki et al (2011) showed that cld-1 is expressed at the membrane in a discontinuous pattern of staining in irradiated compared to unirradiated cells. This dislocation of cld-1 was also accompanied by a reduction in TEER (Yuki et al.,
2011). Interestingly, in the present study, the TEER subsequently rises but not to levels seen in unirradiated cells. This suggests that UV irradiated cells can make TJs but these are unsteady compared to those produced in unirradiated cells.
In cells not yet committed to assembly of junctions i.e. undifferentiated cells in low calcium, irradiation followed by calcium switching leads to development of TJs functionally identical to those in unirradiated cells. This result is in keeping with Western blotting data clearly demonstrating that TJ protein levels remain constant if cells are calcium switched immediately following irradiation. However, if cells are unirradiated and not switched, they go on to loose cld-1 and increase levels of cld-4. Therefore, it is possible that the stimulus provided, by calcium switching, overrides the effects of the
UVR, at least in the short term. However, it is possible that irradiation of undifferentiated cells followed by calcium switching e.g. 24-hours later, may yield different functional effects. These possibilities will be investigated in future studies.
In conclusion, the data presented in this chapter demonstrate that the effects of
UVR on TJ protein expression is dependent on the differentiation state of keratinocytes and is in good agreement with observations made in human tissue. Furthermore, UVR has functional effects which results in decreased TJ barrier function.
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7. GENERAL CONCLUSIONS AND FURTHER WORK
In this study, the first of its kind in human skin, the effects of ageing on TJ protein expression have been investigated. Furthermore, both chronological (intrinsic) and photo ageing (extrinsic) have been studied.
Previous studies in rodent models have demonstrated that TJ permeability and expression of proteins alters with chronological age in a number of tissues (D'Souza et al.,
2009). A gene expression study conducted in house comparing TJ message levels in aged skin vs young skin also suggested that TJ gene expression may be altered in ageing.
However, the results of the study described in chapter 3 clearly demonstrate that there are no significant differences in the expression of TJ proteins in aged vs. young human skin.
This is a surprising given the studies in other tissues and also works in skin showing that barrier function changes with age. However, two things must be noted:
1) No functional work was conducted in the present study so it is not possible to be
absolutely certain that TJ function does not change in age. However, it is possible
to be relatively certain that TJ protein levels and morphology do not change in
chronological ageing in human skin.
2) The studies showing changes to barrier function in aged vs young skin are
generally conducted on what could be very photoexposed anatomical locations e.g.
volar forearm. Therefore, it is not possible to be sure that the effects observed in
these studies are not due to UVR (Ghadially, 1995).
As shown in chapter 4, chronic exposure to UVR results in profound changes to TJ protein expression. These changes include down regulation of cld-1 and upregulation of 137
clds-7 and -12. The overall change to the TJ protein expression profile in photoaged skin is consistent with a decrease in barrier function given what is currently known about the contribution of individual cld isoforms to TJ barrier function. Indeed, such a decrease in TJ barrier function was observed in response to UVB in cultured keratinocytes (section 6.2.5) although of course care must be taken in extrapolating from cells in culture to the in vivo situation. Nevertheless the data suggest strongly that chronic exposure to UVR may be associated with a loss of TJ barrier function. At least some of the changes to the skin permeability barrier observed in other studies (e.g. Lim et al., 2007) may therefore, be attributed to aberrant TJ protein expression although further work will be required to demonstrate this.
Chapter 5 investigated the effects of a single dose of UVB on TJ protein expression in human volunteers. This study was limited because of availability of tissues and so only those proteins observed to have changed expression in chronic photoageing were studied.
Interestingly, the same changes observed in chronic photoageing were also observed in these proteins following a single dose of UVB although some of these changes (increased clds-7 and -12) were not significant. However, the reduction in cld-1 was significant within
24 hours of a single dose of UVB. Thus, this work suggests that the changes in TJ protein expression observed in photoaged skin, may be set in motion at least to some extent by a single dose. Since it was not possible to take multiple biopsies over time following UVR, it is not known whether the increases in clds 7 and 12 may have become significant at later time points. Alternatively, skin may need multiple doses of UVR to achieve the claudin profile observed in photoaged skin. Furthermore, the data suggests that the changes observed in chronic photoageing are indeed as a result of UVR (as opposed to other
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environmental stimuli) since most of the changes can be recapitulated by a single dose of
UVR.
Interestingly, much of the loss of cld-1 expression was associated with the basal layer of the epidermis. Although it was not possible to investigate recovery over time in human skin, work in keratinocytes suggests that cld-1 expression may not recover very quickly with time (fig 6.3). Furthermore, UVB appears to exert its effects on cld-1 expression only in undifferentiated keratinocytes which are phenotypically more like the basal layer of the epidermis. In the in vivo situation cld-1 levels are significantly reduced following a single dose of UVB (fig 5.1) and it is highly likely that in daily life, an individual would, within a 24 hour period, be exposed to multiple doses of UVB.
Therefore, given that cld-1 recovery at least in keratinocytes is slow; this raises the interesting prospect that the loss of cld observed following an acute dose may explain the reduced levels also observed in photoaged skin. Since it appears to be basal cells that experience most reductions in cld expression, this has led to the following hypothesis:
Cells in the basal layer loose cld-1 in response to UVB. Whilst migrating to the upper layers, they will experience further doses of UVB which leads to further downregulation of cld-1. By the time the cells arrive in the granular layer, cld-1 levels are reduced to the levels observed in chronically photoaged skin.
However, at the same time as cells are experiencing reductions in cld levels due to
UVR, there must be some repair/protective mechanisms at work. One such mechanism may be the calcium gradient across the epidermis. This is suggested by two lines of evidence:
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1) Loss of cld in whole human skin is not as significant in the upper layers of the
epidermis where the calcium concentrations are highest
2) Keratinocytes exposed to high levels of extracellular calcium are not subject to
reductions in cld-1 in response to UVB (fig 6.6).
In addition, further work by others in the laboratory also suggests that cld-1 levels may be modulated by the molecule, cis-urocanicacid(O’Neilland Gibbs, 2011). This is made from the chromophore, trans-urocanic acid, in a photoisomerisation reaction. Little is known regarding the role(s) of urocanic acid in skin but it is present in high levels. Work in keratinocytes has demonstrated that cis, but not trans-urocanic acid, increases cld-1 message (Kaneko et al.,2008)andprotein levels (O’Neill and Gibbs, 2011).Therefore, when UVR interacts with skin, cld-1 levels are reduced and at the same time potentially raised in response to the production of cis urocanic acid. It will be the balance of all the different factors involved in modulating cld levels (cis urocanic acid, calcium gradient and possibly others) that ultimately produces the pattern of cld expression observed in chronic photoageing.
Claudin-1 is known to be critical to the barrier function of skin in man (De Benedetto et al., 2011; Hadj-Rabia et al., 2004) and mouse (Furuse et al., 2002). Therefore, the results from all these studies suggest that loss of cld-1 in acute and chronic photoexposure may lead to a loss of barrier function. However, clearly functional data in human skin will be required before firm conclusions can be made.
At present the relevance of cld-1 expression throughout all the living layers of the epidermis is not known since the TJ complexes themselves are specifically located in the granular layer. Therefore, cld-1 and loss of its expression may also be responsible for
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other changes associated with chronic exposure to UVR. For example, photoaged skin is known to be thickened and leathery in appearance due to keratinocyte hyperproliferation.
Interestingly, genetic silencing of cld-1 in primary human keratinocytes also results in hyperproliferation. Hence, it is possible that the role of cld-1 may extend to other functions besides barrier function. Chronic UVB downregulation of cld-1 may therefore, also have other consequences for skin.
The mechanisms by which changes to TJ protein expression occur in response to
UVR are at present not certain. However, in photoaged skin, changes to gene transcription are a likely mechanism because the levels of cld-1 and cld-12 message were altered in the same direction as the protein. Interestingly, in acute UVR irradiated keratinocytes, no change to message levels was observed. This might suggest that acute UVR doses do not alter transcription of TJ proteins. However, tissue limitations prevented qPCR from being carried out on acutely irradiated skin so no change to message levels may be a phenomenon only seen in keratinocytes. On the other hand, the data might suggest that acute, single doses of UVR do not affect transcription, but the multiple doses and differing doses experienced physiologically in in vivo skin over a lifetime, may alter transcriptional levels of specific TJ genes.
In summary, the data presented in this thesis demonstrate several new findings:
1) TJ protein expression does not change in chronologically aged skin
2) TJ proteins in photoaged human skin are aberrantly expressed
3) Acute UVR induces significant changes to TJ protein expression similar to
those seen in photoaged skin
141
4) In keratinocytes exposed to doses of UVR significant changes to TJ protein
expression and function are observed, however, no recovery in the expression
levels of TJ proteins was observed over time post irradiation.
Strengths and Weaknesses -Further Work
The major strength of this study is that it has been conducted for the most part, in human tissue. This is unlike most other studies on either TJ in ageing or the effects of
UVR which have been largely conducted in mice or cell culture models. Hence, the results of the present study are highly relevant to human biology, having been conducted on human volunteers.
The major weakness of the study is the lack of functional data in human skin.
However, at present, functional measurement of TJs in skin is in its infancy. At present there is only one assay which measures the penetration of a dye throughout the epidermis.
This dye is stopped in the granular layer by the presence of the TJ (Furuse et al., 2002).
Although, this assay is now set up within the laboratory, ethical constraints on the tissue availability made it impossible to perform this assay. Hence keratinocyte monolayers were used for this part of the study.
Therefore, an obvious further investigation would be to write a new application for ethical permission to perform the dye penetration assay in aged vs young skin and in photoaged vs photoprotected human skin.
Additional further work on the mechanisms underlying changes to TJ protein/gene expression could also include understanding the role of ROS in the downregulation of TJ proteins. This could be modelled in keratinocytes perhaps using hydrogen peroxide to
142
produce ROS. Additionally, the effects of UVA and UVB and the precise wavelengths involved could be investigated.
Ultimately, understanding how TJs are modified by acute and chronic UVR, and the overall implication for the epidermal barrier could lead to the development of new interventions aimed at preserving TJs. Additionally, understanding how the TJ repairs following UVR may help in the development of therapies aimed at increasing the speed of repair so that the skins barrier is not compromised for long periods of time.
143
APPENDICES
Appendix 1. Preparation of gelatine subbed slides
An adhesive for sections is prepared by mixing dilute aqueous solutions of gelatine and chromium potassium sulphate (chrome alum). Trays of slides are dipped in the mixture for a few seconds. The treated slides are then allowed to air-dry in a dust free place. Slides quated with chrome-gelatinareoftencalled“subbedslides”.Theycanbekeptforatleast three years before using. Cryostat sections can be collected onto the slides and allowed to thaw and dry.
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Appendix 2. Preparations of immunohistochemistry solutions.
Immunohistochemistry Preparation solutions A mixture of 1 phosphate buffered saline tablet and 8g 4% (v/v) paraformaldhyde (PFA) in 200 ml distilled water was stirred Paraformaldhyde and heated for 20 minutes until the PFA was dissolved.
1x Tris Buffered Saline (TBS) A mixture of 5g Tris, 1.2g Na2HPO4, 0.15g NaH2PO4 and 7g NaCl in 1 liter of distilled water.
0.5% (v/v) Triton-X 100 A mixture of 500µl Triton X-100 and 100ml 1X TBS.
Blocking solution: 1% blocking solution: - 50µl sera and 0.050g bovine serum albumin (BSA) were added to 5ml 1X TBS.
3% blocking solution: - 150µl sera and 0.15g bovine serum albumin (BSA) were added to 5ml 1X TBS.
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Appendix 3. Preparations of western blotting solutions.
Western blotting solutions Preparation
APS 10% 1 g Ammonium Persulfate
10 mL distilled H2O Blocking Buffer To 10 mL of T-TBS 1X, add 0.5 g of non fat dry milk.
90.75 g Tris Running Buffer 1.5 M, pH 8.8 500 mL distilled H2O
Adjust the pH at 8.8 with HCl
SDS 10% (w/v) 100 g Sodium Dodecyl Sulfate
1000 mL distilled H2O 6 g Tris Stacking Buffer 0.5 M, pH 6.8 100 mL distilled H2O
Adjust the pH at 6.8 with HCl.
15 g Tris
TANK Buffer 10X 72 g Glycine 50 mL SDS 10% (w/v) Complete to 500 mL with distilled water.
24.2 g Tris 84 g NaCl TBS 10X Adjust the pH to 7.6 with HCl. Complete to 1L with distilled water.
3.03 g Tris HCl 14.4 g Glycine
Transfer buffer 1X 200 mL Methanol 1 mL SDS 10% (w/v) Complete to 1L with distilled water.
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