Heat Shock Proteins in Diabetes and Wound Healing

Current Protein and Peptide Science, 2009, 10, 85-95 85 Heat Shock Proteins in Diabetes and Wound Healing

Mustafa Atalay1,*, Niku Oksala1, 2, Jani Lappalainen1,3, David E. Laaksonen1,4, Chandan K. Sen5 and Sashwati Roy5

1Institute of Biomedicine, Physiology, University of Kuopio, Finland; 2Department of Surgery, Tampere University Hos- pital, Finland; 3Wihuri Research Institute, Helsinki, Finland; 4Institute of Clinical Medicine, Internal Medicine, Univer- sity of Kuopio, Kuopio, Finland; 5Comprehensive Wound Center, Department of Surgery, Dorothy M. Davis Heart & Lung Research Institute, The Ohio State University Medical Center, Columbus, OH, USA

Abstract: The heat shock proteins (HSPs), originally identified as heat-inducible gene products, are a highly conserved family of proteins that respond to a wide variety of stress. Although HSPs are among the most abundant intracellular proteins, they are expressed at low levels under normal physiological conditions, and show marked induction in response to various stressors. HSPs function primarily as molecular chaperones, facilitating the folding of other cellular proteins, preventing protein aggregation, or targeting improperly folded proteins to specific pathways for degradation. By modulating inflammation, wound debris clearance, cell proliferation, migration and collagen synthesis, HSPs are essential for normal wound healing of the skin. In this review, our goal is to discuss the role and clinical implications of HSP with respect to skin wound healing and diabetes. The numerous defects in the function of HSPs associated with diabetes could contribute to the commonly observed complications and delayed wound healing in diabetics. Several physical, pharmacological and genetic approaches may be considered to address HSP-directed therapies both in the laboratory and in the clinics. Keywords: Heat shock proteins, wound healing, tissue protection, diabetes.

INTRODUCTION 1. HSPs: FUNCTIONS AND IMPLICATIONS IN WO- UND HEALING The heat shock proteins (HSPs), originally identified as heat-inducible gene products, are a highly conserved family HSPs are among the most abundant intracellular pro- of proteins (Table 1) that respond to a wide variety of stress. teins. Although expressed at low levels under normal physio- HSPs protect against tissue injury by maintaining synthesis logical conditions, HSPs show dramatically increased ex- and proper conformation of proteins, repairing damaged pro- pression in response to cellular stress. HSPs function primar- teins, and promoting the healing of injured tissue. The syn- ily as molecular chaperones, facilitating the folding of other thesis of HSPs can be induced by either physical or pharma- cellular proteins, preventing protein aggregation, or targeting cological inducers, resulting in cytoprotection against a sub- improperly folded proteins to specific pathways for degrada- sequent potentially irreversible injury [1]. tion [4-6]. HSPs improve cellular survival by repairing denatured proteins, dissociating initial loose protein aggregates Barrier function represents a major role of skin. Wound- and ensuring correct folding and translocation of proteins ing induces HSPs, particularly in the epidermis [2]. In the [7]. This function is also needed in physiological conditions initial phase of wound healing there is an inflammatory re- during de novo protein synthesis, folding of nascent polypep- sponse, followed by organization of the fibrin-rich exudates tides and transport [8]. In case of severe damage, HSPs di- and subsequent re-epithelialization and formation of granula- rect damaged proteins for degradation within the proteasome tion tissue. Complicated by ischemia, infection and related system [4-6]. HSPs play a key role in facilitating immune factors the wound site represents a hostile environment for responses, because they can bind antigenic peptides and the cells involved in the repair process. The wound bed con- transport them to antigen-presenting cells and T lymphocytes tains abundant inducible HSP70 which contributes to protein [9]. Initially, HSPs were thought to be only functional in the homeostasis and cell survival within the healing wound [3]. cytoplasm and nucleus. However, recently they have been HSP functions are compromised under conditions of diabe- implicated in intercellular signaling and transport after re- tes. Here, our goal is to discuss the role and clinical implica- lease to extracellular space and to bloodstream [10-12] (Ta- tions of HSP with respect to skin wound healing and diabe- ble 1). HSPs are also capable of binding to adjacent cells, tes. In addition, we posit mechanisms which may lead to initiating signal transduction [11,13]. The heat shock re- impaired HSP defenses and wound healing in diabetics. sponse attenuates pro-inflammatory mechanisms and inducible nitric oxide synthase activity. It also stabilizes IB, which inhibits NF-B-dependent transcription [14]. Re- *Address correspondence to this author at the Institute of Biomedicine, cently, it has been recognized that HSPs play a dual role in Physiology, University of Kuopio, P.O. Box 1627, FI-70211, Kuopio, Fin- regulating immune responses [15]. Indeed, while intracellu- land; Tel: +358-163108; Fax: +358-163112, E-mail: [email protected] lar HSP induction in response to pro-inflammatory stimuli

Table. 1. Current Mouse HSP Nomenclature. HSP names based on Mouse Genome Informatics database (MGI 3.54, updated January 05, 2008), The Jackson Laboratory

Current Name Gene Additional information Symbols

HSP110 Hsp110 Aka: HSP-E7I, HSP105, HSP110. Cytoplasm, nucleus. Protein domain: HSP70

HSP90kDa, member1 Hsp90b1 Aka: endoplasmin, ERp99, gp96, GRP94, Targ2, Tra-1, Tra1, tumor rejection antigen (gp96) 1. Endoplasmic reticulum, extracellular space. Protein domains: HSP90, endoplasmin.

HSP90kDa Hsp90ab1 Aka: 90kDa, C81438, HSP84, HSP84-1, HSP90, HSPcb. Cytoplasm, mitochondria. Protein class B, member 1 domain: HSP90.

HSP90kDa Hsp90aa1 Aka: 86kDa, 89kDa, hSP4, HSP86-1, HSP89, HSP90, HSPca. Cytoplasm. Protein domain: class A, member 1 HSP90.

HSP70, ps1 Hsp70-ps1 Aka: 70kDa.

HSP34 Hsp34 Aka: 34kDa, p34.

HSP25, ps1 Hsp25-ps1 Aka: 25kDa, ENSMUSG00000007852.

HSP14 Hspa14 Aka: 70kDa, HSP70-4, HSP70L1, NST-1. Protein domain: HSP70

HSP12B Hspa12b Aka: 2700081N06Rik. Protein domain: HSP70

HSP12A Hspa12a Aka: 1700063D12Rik, HSPa12a, mKIAA0417. Protein domain: HSP70

HSP 86 ps1-4 Hsp86-ps1-4 ps1 aka: 86kDa, Hsp86-2, Hsp90. ps2 aka: 86kDa, Hsp86-3, Hsp90. ps3 & ps4 aka: 86kDa.

HSP9 Hspa9 Aka: C3H-specific antigen, CSA, GRP75, Hsc74, HSP74, HSP74a, Hspa9a, mortalin, mot-2, mthsp70, PBP74. Cytoplasmic, mitochondrial. Protein Domains: HSP70, chaperone DnaK, calcium-binding EF-hand

HSP9, ps1-2 Hspa9-ps1-2 ps1 aka 70kDa, Hsc74-ps1, HSP74-ps1, HSP74b, HSPa9b; ps2 aka: Hsc74-ps2

HSP, -crystallin related B9 Hspb9 Protein domain: HSP20

HSP8 Hspa8 Aka: 2410008N15Rik, 70kDa, Hsc70, Hsc71, Hsc73, HSP73, Hspa10. Cytoplasmic, Nuclear. Protein domain: HSP70

HSP8 Hspb8 Aka: Cryac, D5Ucla4, E2IG1, H11, H11K, HSP20-like, HSP22

HSP7 (CV) Hspb7 Aka: 27kDa, cvHsp, HSP25-2. Cytoskeletal

HSP, -crystallin related B6 Hspb6 Aka: HSP20. Eye lens

HSP5 Hspa5 Aka: 78kDa, Bip, D2Wsu141e, D2Wsu17e, Grp78, Hsce70, mBiP, Sez7. Endoplasmic reticulum.

HSP4-like Hspa4l Aka: 94kDa, APG-1, OSP94. Cytoplasm, nucleus. Protein domain: HSP70

HSP4 Hspa4 Aka: 70kDa, APG-2, Hsp110, Hsp70RY. Cytoplasm. Protein domain: HSP70

HSP,3 Hsp84-3 Aka: 84kDa, HSP3, HSP90.

HSP,2 Hsp84-2 Aka: 84kDa, hsp2, Hsp90.

HSP3 Hspb3 Aka: 2310035K17Rik, 27kDa, spb3. Protein domain: HSP20

HSP,2 ostu Hsp70-2os Chromosome 12

HSP2 Hspa2 Aka: 70kDa, Hsp70-2. Mitochondria. Protein domain: HSP70

HSP2 Hspb2 Aka: 27kDa, 2810021G24Rik, HSP27, MKBP. Soluble fraction. Protein domain: HSP20

HSP1A Hspa1a Aka: Hsp70-3, Hsp70.3. Cell survival, DNA repair

HSP1B Hspa1b Aka: HSP68, HSP70, HSP70-1, HSP70.1, HSP70A1. Cell survival, DNA repair

HSP1-like Hspa1l Aka: 70kDa, Hsc70t, Msh5. Protein domain: HSP70

HSP1 Hspb1 Aka: HSP25. Cytoplasm, contractile fiber. Protein domain: HSP20 Heat Shock Proteins in Diabetes and Wound Healing Current Protein and Peptide Science, 2009, Vol. 10, No. 1 87

(Table 1) contd….

Current Name Gene Additional information Symbols

HSP1 (Cpn) Hspd1 (HSP60) Mitochondria

Hspe1 Mitochondria (Mt Cpn 10)

Hspe1-ps1-6 HSP1 pseudogenes

Hspe1-rs1 10kDa, Cpn10-rs1, EPF

DnaJ (HSP40) homolog, subfamily Dnaja1 Old name: HSP DNAJ-like 2. Membrane. Protein domain: HSP DnaJ A, member 1

DnaJ (Hsp40) homolog, subfamily Dnaja1-ps Old name: HSP DNAJ-like 2, pseudogene. Aka: Hsj2-ps. A, member 1, pseudogene

DnaJ (Hsp40) homolog, subfamily Dnaja4 Old name: HSP DNAJ-like 4. Membrane. A, member 4

DnaJ (Hsp40) homolog, subfamily Dnajb3 Old name: HSP DNAJ-like 3. Aka: Hsj3, MSJ-1, Msj1.Protein domain: HSP40. B, member 3

DnaJ (Hsp40) homolog, subfamily Dnajc4 Old name: heat shock 40kD protein 2. Aka: 2010301J22Rik, Hspf2, Mcg18. Membrane, C, member 4 extracellular space.

Serine (or cysteine) peptidase in- Serpinh1 Old name: heat shock protein 47 kDa. Endoplasmic reticulum, extracellular space. hibitor, clade H, member 1

Transmembrane protein 132A Tmem132a Old name: heat shock protein 5 binding protein 1. Aka: 6720481D13Rik, Hspa5bp1. Endo- plasmic reticulum, golgi apparatus.

AHA1, activator of heat shock Ahsa1 Aka: MGC:36589, MGC:36618, p38. Cytoplasm, endoplasmic reticulum. protein ATPase homolog 1 (yeast)

AHA1, activator of heat shock Ahsa2 Aka: 1110064P04Rik. protein ATPase homolog 2 (yeast)

human ortholog name: HSPB (heat Hspbap1 Aka: 3830421G21Rik. Protein domains: Protein Associated with Small Stress protein 1 shock 27kDa) associated protein 1 (PASS1); Transcription factor jumonji/aspartyl -hydroxylase.

Cpn, Chaperonin; EPF, early pregnancy factor; Aka, also known as; CV, cardiovascular; ps, pseudogene; ostu, opposite strand transcription unit. can exert anti-inflammatory effects, extracellular HSPs may most abundant proteins in other tissues, it is not present in signal danger, activating immune cells [15]. Because of the large amounts in the normal skin [20]. Nevertheless, HSP90 overlap in their functions, HSPs have been classified into is constitutively expressed in the superficial epidermis in the families according to their rough molecular weight and ho- suprabasal keratinocytes [2, 21] and has been shown to be mology, i.e. HSP100, HSP90, HSP70, HSP60, HSP40 and induced by heat stress [22] and skin wounding in the superfi- small HSPs. cial thickened epidermis [21]. The presence of HSP90 in the suprabasal layers during wound healing suggests a role in a. HSP90 keratinocyte differentiation [21]. Wound hypoxia, a consequence of alterations in systemic hemodynamics and vascu- The HSP90 family comprises of HSP90 and HSP90, lar disruption associated with wounding, promotes fibroblast which form an inactive complex with the steroid hormone migration especially in the absence of growth factors via a receptor before agonist binding and therefore participate in mechanism dependent on hypoxia-inducible factor-1alpha steroid hormone signaling [16]. HSP90 is responsible for (HIF-1) -mediated secretion of HSP90 into the extracellu- catalyzing the interaction with several substrate proteins and lar space [23]. This HIF-1-mediated pathway also controls co-chaperones involved in cell regulation and intracellular the secretion of multiple growth factors complementing the signaling [17]. HSP90 is a potent autoantigen and thought to another pathway dependent on signaling via reactive oxygen have a role in various inflammatory diseases including arte- species [24, 25]. Interestingly, application of purified recom- riosclerosis [18]. HSP90 also plays an important role in the binant HSP90 protein within carboxymethylcellulose activation of endothelial nitric oxide synthase (eNOS) result- cream enhances skin wound healing during days 5-13, with ing in increased synthesis of vasoregulatory NO and con- an overall improvement of 30% in the time needed for comitant reduction of the eNOS-derived radical, the superox- wound healing [23]. ide anion [19]. Despite the fact that HSP90 is one of the 88 Current Protein and Peptide Science, 2009, Vol. 10, No. 1 Atalay et al. b. HSP70 family lial cells are rich in HSP72 [44]. Of note, there is an inverse correlation between HSP72 levels and the latency of the The HSP70 family comprises eight isoforms, namely healing process, and HSP72 and essential wound healing- three different forms of HSP72, HSPA2, Grp78, HSP70B, related growth factors are expressed in a co-ordinated man- HSP73 and Grp75 [26,27]. The isoforms of HSP are located ner during the initial phase of healing at 7-14 days [42]. mainly in the cytosol and nucleus, but they have also been HSP72 is passively released after cell death or plasma mem- detected in the lysosomes and endoplasmatic reticulum [26]. brane wounding but also actively secreted via the exosome Grp75 has also been detected in the mitochondria [27]. system [10, 45, 46]. Subcutaneous HSP72 delivery into mur- HSP72 is the major inducible HSP found in the nucleus and ine wounds accelerates the wound closure in a dose- cytosol [28]. HSP72 requires ATP for its chaperone activity dependent manner [47]. This effect was attributed to im- [8] and minimizes aggregation of newly synthesized pro- proved clearance of wound debris by enhanced macrophage- teins. Stress-induced HSP72 is effective against aggregation dependent phagocytosis [47]. After skin wounding there are of denatured proteins. Moreover, HSP72 is also capable of significant differences in the expression pattern of different inhibiting stress induced apoptosis [29], even after the acti- HSPs [48]. Whereas HSP72 and HSP32 are expressed only vation of effector caspases [30]. Interestingly, HSP72 is lo- in the epidermis, HSP47 is expressed in both the epidermis cated on the luminal side of the lysosomes where it stabilizes and dermis, and only after skin wounding has occurred [48]. the lysosomal membrane and inhibits the release of hydro- Whether exogenously applied HSP72 may stimulate wound lases and subsequent mediators of cell death [31]. Both ex- re-epithelialization remains to be proven. cessive or impaired synthesis of HSP72 may result in disturbances of cell proliferation [32]. Overexpression of HSP72 also increases the survival and reduces oxidative damage of c. HSP70 and HSF-1 murine fibroblasts against ultraviolet light and suppresses the The regulation of HSP synthesis is controlled mainly by a inflammatory response [33]. Interestingly, in these experi- major transcription factor heat shock factor-1 (HSF-1) which ments, the effect of HSP72 seemed not to be mediated by binds to the heat shock elements (HSE) present in promoter antagonism of ultraviolet light-induced reactive oxygen radi- region of specific genes. Posttranscriptional mechanisms are cals [33]. The role of HSP73 is controversial. It seems to act also implicated in the regulation of HSP synthesis [49]. Un- as a chaperone in non-stressed cells [1] and it is also likely to der physiological conditions, HSF-1 monomers are co- have a role in the formation of clathrin and several other localized with HSP70 in the nucleus. HSF-1 is activated by factors involved in intracellular transportation [34]. cellular stress [49]. The activation process involves trimeri- HSPs may be viewed as survival proteins possessing an zation of HSF-1 monomers, translocation of the trimers, hy- intrinsic ability to confer protection against most apoptotic perphosphorylation and binding to the promoter of heat stimuli [35-37]. Both HSP70 (HSP72) and the heat-shock shock genes [50-52]. The end-products of this process, such cognate protein70 (HSC70, HSP73) are capable of inhibiting as HSP70, exert negative feedback regulation [53]. The post- apoptosis by inhibiting release of cytochrome c, processing transcriptional mechanism involves stabilization of HSP70 of pro-caspase-9 and activation of initiator caspases [29]. mRNA [54]. HSF-1 and nuclear factor-B (NF-B) signal- Heat stress results in inhibition of effector caspases upon ing are tightly linked because in certain conditions, HSF-1 subsequent heat shock [29], and thereby confers protection inhibits NF-B activation [55], Fig. (1). In addition to pro- against heat-induced apoptosis. HSP70 is capable of render- tein denaturation, stress signals may also originate from cell ing cells resistant to cell death induced by TNF- [36] as membranes. Recently it has been proposed that the lipid well as by caspase-3 overexpression [30]. composition and the architecture of membranes act as membrane sensors and modulate HSP response through the acti- HSP72 is constitutively expressed in keratinocytes, espe- vation of HSF-1 [56]. cially in the upper layer of the epidermis [38-40] and also in fibroblasts, macrophages and endothelium [41]. HSP72 has d. HSP60 been proposed to have a role in the wound healing process, which is supported by the finding that its expression is rap- The HSP60 family is made up of only HSP60 itself idly induced after skin wounding in animal models [42]. which is also stress inducible [28]. In contrast to HSP70, Polymer implants (polypropylene, polyester, polypropylene HSP60 is present in both mitochondria and the cytosol. HSP polyglactin) intended to be used in the repair of large ab- 60 has a complicated role in innate immune response [57] dominal wounds induce expression of HSP70 predominantly and in mitochondrial protein biogenesis [58, 59]. HSP60 in macrophages and local inflammatory response, dependent may inhibit caspase-3 [60] or facilitate the maturation of pro- on the material [43]. Interestingly HSP70 expression shows caspase-3 to its active form [61]. The capacity of HSP60 to an inverse correlation with the local inflammatory response stabilize mitochondrial proteins, promote mitochondrial pro- [43]. The anti-inflammatory and pro-proliferative effects of tein biosynthesis and prevent induction of mitochondrial HSP72 support its potential role in wound healing [21, 42, apoptosis seems to be crucial for its cytoprotective function 44]. [62]. HSP60 is constitutively expressed in skin mainly in the epidermis [2, 21] and is induced by skin wounding in the In chronic wounds, such as decubitus ulcers and in post- deep neoepidermis in the basal and suprabasal keratinocytes traumatic wounds, the expression of HSP70 protein has been [21]. It is likely that HSP60 is associated with proliferation noted to be low. In contrast, in healing wounds with rich and migration of keratinocytes in close proximity to the granulation tissue the expression of HSP70 is high [44]. In wound microenvironment [21]. the granulation tissue of healing wounds, only the endothe-

Heat Shock Proteins in Diabetes and Wound Healing Current Protein and Peptide Science, 2009, Vol. 10, No. 1 89

Fig. (1). Schematic presentation of the regulation, mechanisms, inducers and inhibitors of heat shock protein (HSP) synthesis and the effect of HSPs on key factors in wound healing. Physical inducers include heat shock, ischemia-reperfusion, physical exercise, heavy metals, toxins, radiation, UV-light, laser, decreased ATP levels, pH and osmolarity changes. Pharmacological inducers include bimoclomol, geranylgeranylacetone (GGA), -lipoic acid, ansamycins, butyrate, prostaglandins, celastrol, terrecyclin-A, BRX-220, PLA2 and nitric oxide (NO). Solid lines represent stimulation and dashed lines inhibition. Dot lines represent both stimulation and inhibition. HSF, heat shock factor; HSE, heat shock element; NF-B, nuclear factor-B; eEFs, eukaryotic elongation factors; eIFs, eukaryotic translation initiation factors. e. HSP40 Family and Hemeoxygenase (HSP32) tion has been recognized as a sensitive consequence of oxidative stress, and overexpression of HO-1 protects against The HSP40 family is made up of HSP47, which is in- oxidative damage in several cell types [1,70]. volved in the procollagen synthesis [63]. HSP47 closely interacts with HSP70 [64]. HSP47 also has the capability to bind to collagen types I-III [65]. It is expressed in the epi- f. HSP27 and Small HSPs dermal basal cell layer of rat fetal and neonatal skin [66], and HSP27 and B crystalline are members of the small HSP is induced only in the neonatal skin for up to 7 days after that stabilize actin microfilaments [71] and inhibit stress- wounding in the dermis and subcutaneous tissue. Induction induced apoptosis [72]. HSP27 function is modulated by of HSP47 is paralleled by increased synthesis of type I colla- phosphorylation [73]. The overexpression of HSP27 results gen and cellular proliferation in subcutaneous tissue [66]. in promotion of endothelial cell migration [74]. HSP27 is The rat neonatal skin is characterized by wound healing constitutively expressed in the epidermis in the superficial without scar formation in the absence of the HSP47 response layers [21]. During wound healing, HSP27 becomes more [66]. Daily injections of antisense oligonucleotide preventing phosphorylated at the wound edge one day after skin incision HSP47 expression in the rat neonatal skin suppressed the [73]. The expression is also increased in the deep epidermis, accumulation of type I collagen in the skin wound, resulting in the suprabasal keratinocytes. Suprabasal keratinocytes are in minimized scar formation [67]. characterized by low proliferation and high differentiation, The heme oxygenases, i.e. HSP32 (HO-1), although not suggesting a role in migration [21]. Overexpression of functioning as molecular chaperones, catalyze the conversion HSP27 in dermal fibroblasts improves wound contraction. of heme to iron, biliverdin and carbon monoxide and regu- Consistently, wound contraction is compromised if fibro- lates inflammatory and immune responses [68, 69]. HO-1, blasts possess low levels of HSP27 [75]. Increased rapid biliverdin and its catalytic product bilirubin have well- elongation and formation of pseudopods, elongated stress characterized antioxidant properties. Iron regulates the ex- fibers, enhanced attachment of cells to the dishes and en- pression of genes such as nitric oxide synthase. HO-1 induc- hanced migration have been reported in fibroblasts overex- 90 Current Protein and Peptide Science, 2009, Vol. 10, No. 1 Atalay et al. pressing HSP27 [74]. The cellular proliferation rate did not 91], and increased in the kidney and liver of diabetic rats correlate with HSP27 expression, which indicates that the [91, 92]. Expression of HSPs is altered in diabetes in a tissue effects on wound contraction and migration were independ- specific manner because of differing susceptibility of the ent of cell growth [75]. The effect of HSP27 on wound con- tissues to injury [77]. In line with decreased myocardial traction in the same model was shown to be mediated by HSP60 expression, another mitochondrial chaperone, namely mitogen-activated protein kinase (MAPK)- activated protein glucose regulated protein (GRP75), has recently been shown kinase 2/3 (MAPKAPK 2/3) -mediated HSP27 phosphoryla- to be decreased in the mitochondria of heart tissue of dia- tion [73]. HSP27 phosphorylation correlated with both fibro- betic rats [93]. blast-populated collagen lattice model contraction and Of interest in the context of diabetes, the synthesis of wound contraction in rats in vivo [73]. These findings appear HSP60 is closely linked to insulin as the deficiency of insu- to be clinically relevant since wound contraction represents a lin down-regulates HSP60 [90, 94], insulin-like growth fac- significant factor in closing an open wound. In addition to tor-1 (IGF-1) and its receptor (IGF-1R) in the diabetic heart acting as a chaperone, HSP22 i.e. -crystallin, regulates cell [95]. In this context it should be acknowledged that IGF-1 is proliferation, apoptosis, neoplasia, cell motility and cellular known to be cardioprotective [96, 97]. Mitochondrial HSP60 redox state [76]. However, its plausible role in cutaneous increases the amount of functional IGF-1R by inhibiting its wound healing remains unknown. ubiquitination in cardiomyocytes [90, 98] thereby averting proteosome-dependent degradation of the protein. This is 2. DIABETES AND HSP important because lower levels of IGF-1R can potentially Both type 1 and type 2 diabetes are characterized by an limit myocardial defense systems leading to diabetic cardio- increased risk for the development of microvascular and myopathy. In contrast, increased expression of IGF-1 and macrovascular complications. In diabetes, endogenous de- increased IGF-1 signaling in the diabetic kidney causes dia- fense systems are overwhelmed, causing various types of betic nephropathy [99]. Furthermore, it has been hypothe- stress. Uncontrolled oxidative stress represents a characteris- sized that HSP60 transport into the mitochondria might have tic feature of diabetes [77-81]. Among the other important been impaired in diabetic myocardium [98]. Thus, the conditions related to diabetes are dyslipidemia, modification mechanism through which diabetes modulates HSP60 levels of proteins and lipids, and perturbations in the tissue antioxi- seems to be organ-specific. Despite the various protective dant defense network [77-80, 82, 83]. These disturbances are actions of the HSPs, some of these proteins have been asso- exacerbated in diabetes with microvascular complications ciated with disease progression in diabetes. For example, such as nephropathy, retinopathy and neuropathy. The anti- HSP47, a collagen-binding stress protein that has a specific oxidant functions of HSP should therefore prove to be help- role in the intracellular processing of pro-collagen molecules ful in fighting diabetic complications. Indeed, the crucial role during collagen synthesis, plays a pathological role in the of HSPs in diabetes is highlighted by their ability to counter- later stages of diabetic nephropathy and has been associated act denaturation of tissue proteins and facilitate cellular re- with glomerulosclerosis and tubulointerstitial fibrosis in rats pair and defense mechanisms. Of note in this context is the with experimental diabetes [100]. observation that the diabetic state may interfere with the syn- The mechanisms by which diabetes may impair HSP thesis of HSPs [84], utilizing either a transcriptional or post- responses remain sketchy at best. Impaired or deficient insu- translational mechanism [85]. In turn, compromised HSP lin secretion in diabetes may be one mechanism leading to expression may contribute to diabetic complications [86, 87] attenuated protein synthesis, especially related to the acute resulting in a vicious cycle. induction of stress proteins, including HSPs. Insulin regu- a. Experimental and In Vitro Studies lates both the initiation and elongation phases of translation by altering the phosphorylation of eukaryotic translation The effect of diabetes on HSPs is tissue-specific. Under initiation factors (eIF2, eIF2B, eIF3, eIF4B, eIF4E, and conditions of experimental diabetes, reduced expression of eIF4G) and eukaryotic elongation factors (eEF1 and eEF2) HSP72 has been associated with impaired cytoprotective [101]. Generally, EF-1alpha plays a major role in the regula- function and delayed tissue regeneration [41, 88]. On the tion of mRNA translation. In rats, experimental diabetes de- other hand, although heat stress induces HSP72 in diabetic creased the rate of peptide-chain elongation which was asso- rats, it failed to protect the isolated heart against ischemia- ciated with a marked reduction in the amount of elongation reperfusion injury despite insulin treatment [89]. We have factor 2 (EF-2). Insulin therapy reversed the effects of diabe- previously noted that experimental diabetes limits the ex- tes on protein synthesis and increased the total EF-2 content pression of HSP72 in heart, liver and vastus lateralis skeletal of diabetic rats to control values [102]. Recently, it has been muscle [77], and also in the kidney [Atalay et al., unpub- noted that experimental diabetes increases the level of EF- lished observation]. Furthermore, the impaired HSP response 1alpha in rats, which was almost completely reversed after a in the diabetic heart was associated with tissue inflammation ten-day vitamin E supplementation. [103]. EF-1alpha mRNA and overt oxidative stress [77]. expression was found to be rapidly affected by oxidative Our group has also utilized endurance type of exercise stress [104]. Interestingly, in rat cardiac myocytes elevated training as a therapeutic tool to restore HSP synthesis in tis- glucose alone significantly reduced EF-2 phosphorylation sues. Although training induced the activation and expres- [105] which is an essential step involved in extension of the sion of HSF-1 in the skeletal muscle and increased HSP72 polypeptide chain in protein translation. levels in every tissue examined, such favorable induction Impaired HSF-1 activation via up-regulation of glycogen was shown to be blunted in diabetic rats [77]. It has been synthase kinase 3 (GSK-3) represents another important path- shown that HSP60 levels are lowered in the heart [77, 90, Heat Shock Proteins in Diabetes and Wound Healing Current Protein and Peptide Science, 2009, Vol. 10, No. 1 91 way in diabetes. GSK-3, an enzyme initially described as a 3. IMPAIRED WOUND HEALING IN DIABETES AND key regulator of glycogen metabolism, is currently known to THE POSSIBLE ROLE OF HSPs be involved in a diverse array of cell functions, including Diabetes affects approximately 170 million people world- suppression of HSF-1 activity [106]. Limited membrane flu- wide, including 20.8 million in the USA. By the year 2030, idity has been proposed to represent another factor limiting HSP synthesis in diabetics [84]. The physical state and lipid these numbers are expected to double [119]. The diabetic foot ulcer is a major diabetic complication, representing a composition of cellular membranes, subtle alterations of serious medical, social and economic problem all over the membrane fluidity, phase state, and microheterogeneity may world [119]. The cumulative lifetime incidence of diabetic function as membrane sensors to regulate HSP response foot ulcers may be as high as 25%. Foot ulcers precede 84% [56]. Various pathological states, including diabetes are as- of all diabetes-related lower leg amputations [120]. sociated with membrane defects, which can be restored with insulin treatment [107]. A number of factors contribute to wound healing defi- ciencies in individuals with diabetes. Hyperglycemia leads to b. Human Diabetes increased glycosylation of immune cells, such as neutrophils and macrophages by inhibiting their normal function and In human studies, HSP72 levels in peripheral blood leu- predisposing both to chronic inflammation and increased kocytes are considerably lower in type 1 diabetic patients susceptibility to infection. Glycosylation of erythrocytes inc- with polyneuropathy than in healthy volunteers [108]. Simi- reases their rigidity, which may predispose to sludging and larly, intramuscular HSP72 and HO-1 mRNA levels are local ischemia in the microvasculature. Susceptibility to ul- lower in type 2 diabetic patients [109]. A study performed in ceration and impairment of wound healing in diabetes in- twins showed that lower levels of HSP72 mRNA in skeletal creases dramatically when accompanied by diabetic periph- muscle are associated with some markers of insulin resis- eral neuropathy and peripheral vascular disease. These com- tance in patients with type 2 diabetes [87]. However, HSP72 plications predispose to microtrauma, foot deformities and mRNA levels did not correlate with either fasting plasma ischemia [120]. glucose or insulin level in any of the subgroups investigated, [87] suggesting that alterations in HSP mRNA is not simply Wound healing occurs as a cellular response to injury and the consequence of chronically elevated plasma glucose or involves a number of coordinated events including clot for- insulin levels. Nonetheless, it has been shown in diabetic mation, inflammation, re-epithelialization, angiogenesis, gra- rodents that pharmacological induction of HSP72 expression nulation tissue formation, wound contraction, scar formation improves insulin sensitivity [110], providing evidence that and tissue remodeling [121, 122]. Pathophysiological proc- HSP72 is directly involved in the pathogenesis of insulin esses that impair wound healing in diabetes include dec- resistance. It was later demonstrated that skeletal muscle reased or impaired growth factor production [123, 124], an- HSP72 mRNA levels indeed tightly correlate with glucose giogenesis [125], leukocyte function and chemotaxis [126], disposal rate and oxidative capacity of tissues [109]. On the keratinocyte and fibroblast migration and proliferation, func- other hand, using a quantitative proteome analysis, Hojlund tion of epidermal nerves [127], collagen accumulation, re-ep- et al. reported increased levels of HSP90 and GRP78 in the ithelialization, granulation tissue formation [124], and tissue skeletal muscle of diabetic subjects, but no relationship be- remodeling by matrix-metalloproteinases (MMPs) [128]. In tween fasting plasma glucose and HSP90 levels were noted addition, narrowing or occlusion of the blood vessels within [111]. Of interest is the observation that HSP90 plays an the edge of the wound leads to tissue hypoxia and abnormal important role in maintaining the activity of protein kinase B wound healing. Oxidative stress, abnormal nitric oxide pro- (PK-B) [112], which is involved in the regulation of insulin- duction and disturbed redox status may also play a role mediated glucose transport and glycogen synthesis. [121]. Abnormal HSP and EF expression may also contribute to Little is known about the role of HSPs in wound healing the altered protein synthesis in diabetes, in addition to the or in the abnormal wound healing in diabetes. The impor- direct effect of insulin on protein synthesis. A marked in- tance of HSPs in several of the processes involved in wound crease in expression of EF-1alpha and an imbalance with healing and their expression as a protective mechanism sug- other subunits of the EF-1 complex has been reported in gests that they may be important in the response to wounds. skeletal muscle of type 1 type 2 diabetic subjects [113]. The diabetic state results in delayed expression of HSP72 at the protein level, despite mRNA upregulation in the epithe- Both type 1 and type 2 diabetes are well-established risk lial cells and inflammatory cells during the wound healing factors for cardiovascular disease. Firm evidence lends sup- process [40]. On the other hand, HSP70 expression in the port to the contention that immunity to HSP60 and its deri- wound bed in diabetic mice increased after a delay, suggest- ved peptides is involved in type 1 diabetes [114, 115]. ing that the poorly healing, chronically open wound may Moreover, it has been recently suggested that circulating result in a more potent HSP induction than a normally heal- HSP60 may contribute to the cardiovascular pathology asso- ing wound. Similarly, in skin fibroblasts isolated from a pa- ciated with diabetes [116], supporting earlier observations on tient with diabetic nephropathy, the levels of HSP70, HSP60 the association of HSP60 and atherosclerosis [117]. Interest- and HSP27 were increased [129]. In streptozotocin-induced ingly, HSP60 shifts the cytokine secretion profile towards diabetic rats, during wound healing, the levels of HSP72 and the Th2 type via toll-like receptor-2 (TLR2)-dependent sig- HSP73 were roughly 60% lower than in normal control rats naling in human T cells [118], which could have a modifying 7-14 days after the incision [41]. This effect of diabetes effect on the immunological response in the pathogenesis of could be prevented by administration of insulin and normali- type 1 diabetes zation of blood glucose levels [41]. According to these re- 92 Current Protein and Peptide Science, 2009, Vol. 10, No. 1 Atalay et al. sults, it seems clear that the diabetic state interferes with the stress [140]. Alpha lipoic acid (-LA) is a naturally occur- expression of HSP72 and HSP73, but the mechanism re- ring antioxidant precursor which induces HSP60 in rat heart mains unclear [41] [91]. At high doses -LA has been suggested to function as a HSP inducer by activating HSF-1 via increasing disulfide 4. STRATEGIES TO INDUCE HSPs IN DIABETES formation in certain target proteins [141]. Indeed, Strokov et al. [108] observed that -LA administration restored HSP a. Physical Inducers of HSPs levels and NO production and improved neuropathic symp- An increased intracellular pool of aggregating denatured toms in a study of type 1 diabetic patients with neuropathy. proteins is the major inducer of HSP70 accumulation [1]. Proteasome inhibitors, including MG-132, lactacystin HSP expression is upregulated by various physical stresses and bortezomib, are potent inducers of the heat shock re- including heat, ischemia, reperfusion, physical exercise, sponse [142-144]. Proteasome inhibitors are proposed to heavy metals and toxins [4, 77, 130]. Transient exposure of increase the amount of misfolded proteins, leading to HSF-1- the tissue to heat results in release of arachidonic acid from dependent transcription [143,145]. Certain proteasome in- plasma membrane resulting in leukotriene and prostaglandin hibitors induce HSP expression by directly inducing HSPs synthesis [11, 72, 131]. Changes in interstitial fluid osmolar- [146]. Another HSF-1 activator, celastrol results in hyper- ity [132], pH and cellular ATP content can also induce HSPs phosphorylation and transcriptional activation of HSF-1 pos- [133]. Heat shock response and synthesis of growth factors sibly via proteasome inhibition [147]. Terrecyclic acid A is a are interrelated, because transforming growth factor- (TGF- novel HSP inducer leading to HSF-1 activation, the mecha- ) regulates synthesis of HSP70 and HSP90 in cultured nism of which seems to be related to perturbation of the re- chicken embryo cells, whereas fibroblast growth factor dox state and increase of reactive oxygen species [148]. In- (FGF) and epidermal growth factor (EGF) are ineffective terestingly, based on interaction of HSP90 with HSF-1, sev- [134]. In cultured fibroblasts exposed to ultraviolet B irradia- eral inhibitors of HSP90, namely radicicol and the benzoqui- tion, TGF- mediates the upregulation of HSP70 synthesis none ansamycins [149], have been found to stimulate the [135]. Hyperthermia induces expression of HSP70 in the HSF-1 activity and subsequently induce HSP expression. keratinocytes in human skin [38]. Specific drugs designed to co-induce HSP expression, Treatment of skin incisions with 815 nm laser light ac- namely bimoclomol [150] and BRX-220 [151], improve dia- celerates wound closure without prominent scar formation, betic retinopathy, neuropathy, nephropathy, wound healing, along with increased expression of HSP70 in the epidermal cardiac ischemia and insulin resistance in animal models and dermal layers [136]. In addition, exposure of rat skin to [110, 152, 153]. Bimoclomol interacts with HSF-1 prolong- laser irradiation resulted in increased expression of HSP72 a ing the binding to DNA, and it also upregulates HSP72 in the day after the exposure in the stratum spinosum and paral- diabetic wound [152, 154]. Altered lipid composition and leled with reduced thickness of the skin layer [39]. The ele- physical state (fluidity) of biological membranes are decisive vated expression of HSP72 was sustained for up to 7 days factors in the processes of perception and transduction of [39]. stress into a signal that triggers the transcriptional activation of genes encoding stress protein. Bimoclomol and its deriva- b. Pharmacological Inducers of HSPs tives specifically interact with and increase the fluidity of Anti-inflammatory drugs such as indomethacin induce negatively charged membrane lipids [155]. Accordingly, the HSF-1 DNA-binding activity by lowering the temperature HSP-related activity of bimoclomol is highly susceptible to threshold for HSF-1 activation to physiologic temperature the fatty acid composition and fluidity of membrane of target range without activation of HSP gene itself, resulting in in- cells [155]. Because the plasma membrane also acts as an creased protection against lethal conditions [72]. Exposure to important regulatory interface, it has therefore been recently sodium salicylate has similar effects [131]. In tissue injury speculated that the lower HSF-1 and HSP levels in diabetes and various inflammatory states, the release of arachidonic could be the result of compromised membrane fluidity [84]. acid has been shown to hyperphosphorylate HSF-1 in a dose- Metformin, a dimethylbiguanide drug that is widely used dependent manner, and to induce DNA binding activity for the treatment of hyperglycemia in type 2 diabetes, also [131] resulting in HSP gene activation and HSP protein syn- has effects on vascular endothelial function, presumably via thesis. HSF-1 is rapidly activated by synthesis of HSF-1- activation of eNOS [156]. HSP90 has an important role in activating prostaglandins, especially cyclopentenone prosta- the activation of eNOS [157]. Interestingly, metformin has glandins [137] and some cytokines [138]. Exposure of the been shown to enhance endothelial function by AMPK- cell to exogenous phospholipase A2 results in HSF-1 DNA- dependent and HSP90-mediated eNOS activation [156]. Be- binding and modification of HSF-1 similar to that observed cause mitogen-activated protein kinases modulate phos- after heat shock [131]. Exposure to hyperthermia results in phorylation of HSP27, and therefore its activity in actin mi- activation of membrane bound A2 [11]. Similar to indo- crofilament function, MAPK modulators are also potentially methacin and arachidonic acid, phospholipase A2 lowers the useful for HSP27 function [73]. thermal threshold for HSF-1 activation [131]. Nitric oxide (NO) synthesis induces HSP expression. Geranylgeranylacetone (GGA) is an antiulcer drug which Inhibition of NO synthesis limits the expression of HSPs induces gastroprotection, HSF-1 activation and HSP70 gene [158]. Medications that have been associated with improved activation [139]. Short chain fatty acids namely butyrate outcome in diabetes, including HMG CoA reductase inhibi- induce dose dependent increase of HSP25 in rat IEC-18 cells tors [159], angiotensin-converting enzyme (ACE) inhibitors and tolerance against monochloramine induced oxidative [160], and thiazolidinediones [161] all can restore endothe- Heat Shock Proteins in Diabetes and Wound Healing Current Protein and Peptide Science, 2009, Vol. 10, No. 1 93 lial NO synthase function which in turn may induce HSP [15] Chen, Y., Voegeli, T.S., Liu, P.P., Noble, E.G. and Currie, R.W. expression. Physical exercise provides a physiological stimu- (2007) Inflamm. Allergy Drug Targets, 6, 91-100. [16] Srinivasan, G., Post, J.F. and Thompson, E.B. (1997) J. Steroid lus for increased NO production [162] and increased HSP Biochem. Mol. Biol., 60, 1-9. and HSF-1 expression [77,163], thus contributing to the im- [17] Whitesell, L. and Lindquist, S.L. (2005) Nat. Rev. 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Received: January 09, 2008 Revised: February 12, 2008 Accepted: March 12, 2008