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Hemidesmosomes Show Abnormal Association with the Filament Network in Junctional Forms of

James R. McMillan, John A. McGrath, Michael J. Tidman,* and Robin A. J. Eady Department of Pathology, St John’s Institute of Dermatology, UMDS, St Thomas’s Hospital, London, U.K.; *Department of Dermatology, Royal Infirmary of Edinburgh, Edinburgh, U.K.

Junctional epidermolysis bullosa is a group of hereditary respectively. In junctional epidermolysis bullosa with bullous disorders resulting from defects in several hemi- pyloric atresia (α6β4 abnormalities, n ⍧ 3) the values -anchoring filament components. Because were also reduced [41.8% ⍨ 7.0 (p < 0.001) and 44.5% hemidesmosomes are involved not only in - ⍨ 5.7 (p < 0.001), respectively]. In the non-Herlitz group adherence, but also in normal ( 5 mutations, n ⍧ 3) the counts were 66.7% ⍨ anchorage of keratin intermediate filaments to the basal 7.1 (p > 0.05) and 70.5% ⍨ 8.5 (p < 0.05), and in keratinocyte membrane, we questioned whether this from patients with antigen 2 intracellular function of hemidesmosomes was also per- mutations (n ⍧ 3) the counts were 54.3% ⍨ 13.8 (p < 0.01) turbed in junctional epidermolysis bullosa. We used and 57.1% ⍨ 13.9 (p < 0.01). In epidermolysis bullosa quantitative electron microscopic methods to assess cer- simplex associated with mutations the values were tain morphologic features of –keratin 31.9% ⍨ 8.9 (p < 0.001) for keratin intermediate filaments intermediate filaments interactions in skin from normal association and 39.9% ⍨ 7.1 (p < 0.001) for inner plaques. subjects (n ⍧ 11) and from patients with different forms Findings in recessive dystrophic epidermolysis bullosa patients’ skin were indistinguishable from normal control of junctional epidermolysis bullosa (n ⍧ 13). In addition, skin with inner plaques (90.5% ⍨ 2.5) and keratin inter- skin from patients with autosomal recessive epidermolysis mediate filaments attachment (86.3% ⍨ 2.1). These bullosa simplex with plectin defects (n ⍧ 3) or with findings suggest that the molecular abnormalities under- autosomal recessive dystrophic epidermolysis bullosa lying different forms of junctional epidermolysis bullosa (n ⍧ 4) were included as controls. Values were expressed appear to affect certain critical intracellular functions as a percentage of the total number of hemidesmosomes of hemidesmosomes, such as the normal connections counted. In normal skin 83.3% ⍨ 3.3 (SEM) hemidesmo- with keratin intermediate filaments. This may have somes were associated with keratin intermediate filaments important implications for the maintenance of basal and 90.1% ⍨ 1.9 had inner plaques. In Herlitz junctional keratinocyte integrity and resilience in junctional epidermolysis bullosa (laminin 5 abnormalities, n ⍧ 4) epidermolysis bullosa. Key words: / these values were reduced to 45.3% ⍨ 11.5 (p < 0.001; blistering skin disease/electron microscopy. J Invest Dermatol analysis of variance) and 50.3% ⍨ 12.8 (p < 0.001), 110:132–137, 1998

emidesmosomes are junctional complexes distributed The molecular pathology of JEB is complex; this autosomal recessive along the inner or stromal-facing aspect of basal cells disease may result from mutations in any one of six distinct genes of the and other stratified epithelia (Tidman encoding different molecular components of the hemidesmosome- and Eady, 1984; Legan et al, 1992; Borradori and anchoring filament complex (Christiano and Uitto, 1996). Sonnenberg, 1996). A major role of hemidesmosomes Initially, the ‘‘lethal’’ or Herlitz form of JEB was shown to be caused isH in epidermal–dermal adhesion and defective hemidesmosome func- by mutations in each of the three genes of the different polypeptide tion may result in epidermal detachment or blistering at the level of chains comprising the trimeric anchoring filament protein, laminin 5 the lamina lucida of the basement membrane, as seen in the group of (Aberdam et al, 1994; Baudoin et al, 1994; Pulkkinen et al, 1994; hereditary mechanobullous disorders, junctional epidermolysis bullosa Kivirikko et al, 1995). It was then found that mutations in either a (JEB) (Tidman and Eady, 1986; Borradori and Sonnenberg, 1996). laminin 5 gene or the gene for another hemidesmosome-anchoring filament component, the 180 kDa bullous pemphigoid antigen (BP180/ BPAG2/COL17 A1), could result in a nonlethal form of JEB known as the ‘‘generalized atrophic benign’’ form of epidermolysis bullosa Manuscript received August 13, 1997; revised October 7, 1997; accepted for (EB) (Jonkman et al, 1995; McGrath et al, 1995a; McGrath et al, publication October 14, 1997. 1995b). More recently, mutations in the α6 and β4 genes Reprint requests to: Professor R. A. J. Eady, Department of Cell Pathology, St John’s Institute of Dermatology, St Thomas’s Hospital, Lambeth Palace Road, have been shown to give rise to a form of JEB associated with pyloric London SE1 7EH, U.K.. atresia (Vidal et al, 1995; Shimizu et al, 1996; Pulkkinen et al, 1997). Abbreviations: BPAG1, bullous pemphigoid antigen 1; BPAG2, bullous The α6β4 integrin is a hemidesmosome component and is a receptor pemphigoid antigen 2; BP180, 180 kDa bullous pemphigoid antigen; BP230, that binds laminin 5 (Niessen et al, 1994). The β4 integrin subunit has 230 kDa bullous pemphigoid antigen. been implicated in the second major function of hemidesmosomes,

0022-202X/98/$10.50 · Copyright © 1998 by The Society for Investigative Dermatology, Inc. 132 VOL. 110, NO. 2 FEBRUARY 1998 HEMIDESMOSOME–KERATIN FILAMENT INTERACTIONS 133 making connections with the (Spinardi et al, 1993; type VII collagen monoclonal antibody (LH7:2) from I. Leigh (Royal London Mainiero et al, 1995; Spinardi et al, 1995; van der Neut et al, 1996; Hospital, U.K.) (Leigh et al, 1987). Indirect immunofluorescence microscopy Niessen et al, 1997). was performed on skin biopsies. The skin specimens were embedded in OCT The linkage of the keratin intermediate filament network to the compound (Miles Diagnostic, Elkhart, IN) and snap frozen in isopentane cooled by liquid nitrogen. Cryostat sections (5 µm) were collected on gelatin and hemidesmosome and the basal keratinocyte plasma membrane may chrome alum-coated slides and air dried. The sections were used immediately involve several components (Katz et al, 1991). Current evidence or stored until needed in air-tight boxes at –70°C. suggests that at least two distinct proteins are involved in this role. Indirect immunofluorescence was carried out with modifications to the The first is the 230 kDa bullous pemphigoid antigen (BP230) or method described previously (Kennedy et al, 1985). Sections were fixed in cold bullous pemphigoid antigen 1 (BPAG1) (Guo et al, 1995), and the acetone (–20°C) for 10 min and incubated with normal rabbit sera for 5 min second is plectin (Smith et al, 1996). Experimental mice genetically at 37°C. Sections were incubated with primary antibodies, secondary antibodies deficient in BPAG1 have small hemidesmosomes with defective connec- rabbit anti-mouse fluorescein isothiocyanate, diluted in 3% bovine serum tions with keratin filaments (Guo et al, 1995). The basal albumin in 0.1 M Dulbecco’s phosphate-buffered saline for 30 min at 37°C in are intrinsically fragile and can be induced by trauma to rupture a darkened, humidified chamber. The sections were then mounted in 0.2% p-phenylenediamine in phosphate-buffered saline and glycerol and examined through the cytoplasm in a zone just above the hemidesmosomes (Guo with a Nikon Optiphot 2 microscope (Nikon UK, Kingston, U.K.) equipped et al, 1995). Although no human genetic disease has yet been shown for epifluorescence. Controls included normal skin as a positive control and to arise from a mutant BPAG1 gene, a similar epidermal lesion has substituting the primary antibody with phosphate-buffered saline, myeloma been described in an autosomal recessive form of EB simplex caused supernatant, or an irrelevant immunoglobulin isotype, as negative controls. by mutations in plectin, another hemidesmosome plaque component that shares DNA sequence similarities with BPAG1 (Gache et al, 1996; Electron microscopy The skin specimens were cut into small pieces and McLean et al, 1996; Smith et al, 1996). Plectin therefore would placed in half-strength Karnovsky fixative containing 2% formaldehyde and 2.5% glutaraldehyde in 0.04 M sodium cacodylate buffer with 0.05% calcium also seem to be involved in the linkage of keratin filaments to 1 hemidesmosomes. A further hemidesmosome component, HD1, has chloride and 5% sucrose (Eady, 1985). Samples were fixed while on a rotator at room temperature for 4–5 h and then washed in three changes molecular mass, tissue distribution, and probable functional similarities (15 min each) of 0.067 M cacodylate buffer with 0.05% calcium chloride to plectin (Hieda et al, 1992; Smith et al, 1996). and 5% sucrose. Postfixation was in 1.3% osmium (2% stock diluted one Currently, therefore, BPAG1, plectin, HD1, and the β4 subunit of part water to two parts osmium) for 2 h at room temperature. Specimens the integrin α6β4 have all been implicated in the linkage of keratin were dehydrated in a graded ethanol series (15 min each), stained en bloc filaments to hemidesmosomes; however, we are unaware of any in 2.5 or 5% uranyl acetate dissolved in 50% ethanol (1 h), and embedded experimental data specifically examining the role of BP180, not only on in TAAB 812 resin with hard hardener (TAAB, Aldermaston, U.K.) via hemidesmosome assembly but also on the capacity of hemidesmosome propylene oxide (two washes). After two changes of resin the samples were plaques to associate or connect with keratin filaments. Recent data placed in silastic moulds, labeled, and placed in an oven at 60°C for 48 h. from in vitro studies (Baker et al, 1996, 1997) using different cell lines After appropriate orientation of the blocks semi-thin and ultra-thin sections were cut on a Reichert OMU-4 ultramicrotome (Leica, Milton Keynes, have, however, provided evidence that laminin 5 can indirectly mediate U.K.). Semi-thin sections (0.5 µm) were stained with azure II and methylene the association of the keratin cytoskeleton with the hemidesmosome blue (Richardson et al, 1960). Ultra-thin sections (60–90 nm) were collected and influence the assembly and stability of the hemidesmosome plaque. on copper grids and stained in 50% alcoholic uranyl acetate (15 min) and We therefore assessed the hemidesmosomes in the skin of patients lead citrate (5 s) and examined in a JEOL 100 CX electron microscope with different forms of JEB showing defective expression of laminin (Welwyn Garden City, U.K.) with an accelerating voltage of 80 kV. 5, BP180, or α6β4 for evidence of perturbed hemidesmosome–keratin filament interactions. Evidence for impaired interactions would have Hemidesmosome analysis Our group has previously analyzed hemidesmo- implications not only for the normal anchorage of the cytoskeleton to some structure and numbers in human skin (Tidman and Eady, 1984, 1986). the stromal face of basal keratinocytes but also for the normal We have employed similar methods in this study to those previously described. Electron micrographs of the dermal–epidermal junction were maintenance of cell integrity. obtained at a standard magnification setting on the electron microscope of ϫ13,000. A carbon diffraction grating with 2160 lines per mm was used MATERIALS AND METHODS for calibration purposes and to calculate the exact final magnification. The Skin samples Skin biopsy specimens were obtained from patients with micrographs were printed with a constant enlargement of ϫ2.6 giving a different forms of EB, either as a routine diagnostic procedure or as part of a final magnification of about ϫ33,800. Areas that contained melanocytes, research project, with the approval of the appropriate research ethics committee. obliquely sectioned dermal–epidermal junction, and basement membrane In all cases, the biopsies were performed with the patient’s or guardian’s reduplication were excluded from this study. For the purposes of standardiza- informed consent. A total of 12 EB patients were included: four with the tion all assessments of hemidesmosome number and structural features were Herlitz form of JEB (age 12 d–4 mo), six with non-Herlitz (nonlethal) JEB performed by one observer (J.R.M.). At least 50 hemidesmosomes were (age 6–60 y), and three with JEB associated with pyloric atresia (age 3 d–3 y). counted for each individual. The basement membrane beneath a minimum Controls included 11 normal individuals without skin disease (age 20–60 y), four of five basal keratinocytes was surveyed, which produced maximum counts patients with recessive dystrophic EB (age 1–14 y) in which hemidesmosomes are of 250 hemidesmosomes in normal skin. In JEB skin, however, there is a morphologically normal (Tidman and Eady, 1985), and three patients with known reduction in the number of hemidesmosomes that led to lower autosomal recessive EB simplex with muscular dystrophy in which hemidesmo- counts. A hemidesmosome was defined as an area of electron dense material some development is known to be impaired by defects in plectin (McLean overlying the cytoplasmic side of the basal keratinocyte plasma membrane. et al, 1996; Smith et al, 1996) (age 3–10 y). Continuous and overlapping pictures of dermal–epidermal junction were The clinical diagnosis of EB was made in each case by at least one of the prepared and all nonobliquely sectioned hemidesmosomes were counted. authors and the diagnosis was confirmed in our laboratory by transmission Each hemidesmosome was scored for the presence or absence of an inner electron microscopy and indirect immunofluorescence microscopy (see below). plaque and for contact of keratin intermediate filament bundles with the Five JEB cases were included in a previous study (Tidman and Eady, 1986). inner plaque (Fig 1). The percentage of hemidesmosomes with inner Results of mutational analysis were available in three patients with laminin 5 plaques and apparent connections with keratin intermediate filaments was defects, two patients with BP180 abnormalities, one patient with altered type VII then calculated. collagen immunostaining, and three patients with abnormal plectin expression. Statistical analysis The data were pooled into seven categories (according to the clinical diagnosis, immunohistochemical, and molecular analysis findings) Indirect immunofluorescence The subtype of EB was confirmed by and the mean and standard error of the mean was calculated for each group. indirect immunofluorescence using a range of monoclonal antibodies specifically Statistical analysis was performed using Minitab (State College, PA) and GLIM reacting with different hemidesmosome-anchoring filament antigens. The 4 software (Frances et al, 1993). One-way analysis of variance was performed following monoclonal antibodies were used: anti-laminin 5 (GB3) from Serotec between the control group and the disease groups. Logistic regression was used (Oxford, U.K.) (Heagerty et al, 1986; Hsi and Yeh, 1986; Matsui et al, 1995); anti-BP180 monoclonal (HD4–233) a gift from K. Owaribe (Nagoya University, Japan) (Nishizawa et al, 1993); anti-integrin β4 antibody (450–11 A) from S. Kennel (Oak Ridge National Laboratory, TN) (Kennel et al, 1990), and anti- 1Karnovsky MJA, J Cell Biol 27:137–138, 1965 (abstr.). 134 McMILLAN ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

the spread of the data for the hemidesmosome–keratin filament association and inner plaque assembly in the BP180 deficient cases was considerably wider than in the laminin 5 deficient cases. Mutational analysis revealed differences in types of BPAG2 mutations affecting the BP180 deficient patients (Table II). One individual with two nonsense mutations showed the lowest values for hemidesmosome inner plaques and keratin filament association within that group (Table II, case 1, and Fig 2d), whereas one patient with a nonsense/missense combination of mutations showed higher values for each ultrastructural feature (Table II, case 2, and Fig 2e). JEB associated with pyloric atresia (Fig 2f) showed a greater disruption of hemidesmosome–keratin filament association and inner plaque assembly than in Herlitz JEB skin (Fig 2b), even though the Figure 1. Schematic representation of a normal hemidesmosome– morphometric data were not significantly different (Table I). This keratin filament complex as observed using transmission electron disruption was not a generalized observation in EB as shown by microscopy. Keratin intermediate filaments are closely associated with the the normal hemidesmosome–keratin filament interactions in severe inner plaque of the hemidesmosome which is parallel to the slightly larger outer recessive dystrophic EB skin (Fig 2h, Table I). Skin from patients plaque overlying the plasma membrane. Below the outer plaque fine anchoring with autosomal recessive EB simplex with plectin mutations showed filaments traverse the lamina lucida towards the lamina densa. The sub-basal dense plate is extracellular and just beneath the outer plaque. the lowest values for hemidesmosome–keratin intermediate filament interactions and inner plaque assembly (Table I, Fig 2g). There were normal desmosome–keratin filament associations in all JEB skin samples, Table I. Morphometric analysis of hemidesmosomes with keratin filament attachment in control and epidermolysis including those from the Herlitz group (Fig 2i). bullosa skin DISCUSSION b Hemidesmosome counts (%) Our findings indicate that primary defects of hemidesmosome- associated proteins known to underlie different forms of JEB are Number of Inner Keratin filament a likely to have a greater effect on the function of hemidesmosomes Type of skin sample samples plaques attachment beyond mere disruption of their attachment to the extracellular matrix. α β Normal control 11 90.1 Ϯ 1.9 83.3 Ϯ 3.3 Our data show unequivocally that defective 6 4 expression, as seen Herlitz JEB 4 50.3 Ϯ 12.8c 45.3 Ϯ 11.5c in the skin of three patients with the form of JEB combined with Non-Herlitz JEB (LN-5) 3 70.5 Ϯ 8.5e 66.7 Ϯ 7.1f pyloric atresia, is associated not only with a highly significant reduction Non-Herlitz JEB (BP180) 3 57.1 Ϯ 13.9d 54.3 Ϯ 13.5d in the number of hemidesmosomes with inner plaques, compared with Pyloric atresia-JEB 3 44.5 Ϯ 5.7c 41.8 Ϯ 7.0c normal controls, but also with morphologic evidence of impaired Recessive dystrophic EB 4 90.5 Ϯ 2.5f 86.3 Ϯ 2.1f keratin filament attachment to these altered hemidesmosomes. Different c c Autosomal recessive EBS 3 39.9 Ϯ 7.1 31.9 Ϯ 8.9 lines of evidence indicate the importance of the α6β4 integrin in (MD-EBS) hemidesmosome assembly and stabilization (Jones et al, 1991; Kurpakus β aJEB, junctional epidermolysis bullosa; EB, epidermolysis bullosa; MD-EBS, epidermo- et al, 1991; Baker et al, 1996). The cytoplasmic tail of the 4 subunit lysis bullosa simplex associated with muscular dystrophy. contains over 1000 amino acid residues and may therefore extend into bAll values are a mean percentage of hemidesmosomes Ϯ SEM. the inner plaque region from the transmembrane domain (Spinardi cDifference from normal control, p Ͻ 0.001. d et al, 1993, 1995; Borradori and Sonnenberg, 1996). Some authors Difference from normal control, p Ͻ 0.01. β eDifference from normal control, p Ͻ 0.05. have suggested that the 4 cytoplasmic domain has the capacity to fDifference from normal control, p Ͼ 0.05. interact indirectly with (Spinardi et al, 1993, 1995; Niessen et al, 1997). Alternatively, keratins may link to hemidesmosomes to check that the variance was greater than the sampling variation and we through intermediary molecular interactions, possibly involving BP230 assumed that each group of data was normally distributed. (Guo et al, 1995). Targeted removal of the β4 gene in experimental mice results in a phenotype characterized by widespread epithelial RESULTS detachment from the stroma, and a virtual lack of identifiable hemides- Morphometric data provide evidence for altered association mosomes (Dowling et al, 1996; van der Neut et al, 1996). Normal between hemidesmosomes and keratin intermediate filaments connections of the keratin filament network to the basal surface of the in different forms of JEB Table I shows the percentage of basal cells would therefore be severely compromised (Dowling et al, hemidesmosomes (expressed as mean Ϯ SEM) from each group 1996; van der Neut et al, 1996). Mutations in the human genes for showing inner plaques and association with keratin intermediate the β4orα6 subunits have recently been described (Vidal et al, 1995; filaments. A high percentage of hemidesmosomes in normal control Pulkkinen et al, 1997; Takizawa et al, 1997). In these and other cases skin had well-formed inner plaques and a clear association with keratin of JEB associated with pyloric atresia the hemidesmosomes were intermediate filament bundles (Fig 2a). Fine filaments (anchoring ultrastructurally small, often lacking a normal sub-basal dense plate, filaments) were observed crossing the lamina lucida beneath the but abnormalities of the connections between hemidesmosomes and hemidesmosomes (Fig 2a). keratin filaments were not emphasized (Smith, 1993; Vidal et al, 1995; Herlitz JEB skin samples showed a reduction in the size and number Niessen et al, 1996; Shimizu et al, 1996). None of the reports included of hemidesmosomes (Fig 2b). There was a complete absence of sub- results of morphometric analysis similar to the type of assessment we basal dense plates and a reduction in the number of hemidesmosomes have used, however, and the number of hemidesmosomes examined with inner plaques and keratin filament association (Fig 2b, Table I). in detail may have been lower. Non-Herlitz JEB skin with abnormalities in BP180 or laminin 5 both Patients with abnormalities in laminin 5 also had abnormalities in showed minor reductions in hemidesmosome size and number (Fig 2e,c, hemidesmosome–cytoskeletal connections, even though laminin 5 is Table I, respectively). The association between hemidesmosomes and an extracellular protein located in the lower lamina lucida at the keratin filaments was less in the samples from patients with BP180 interface with the lamina densa (McGrath et al, 1994). Skin biopsies defects (Table I, Fig 2d,e). The differences between the mean values from patients with Herlitz JEB, which is caused by nonsense mutations of the number of inner plaques detected and hemidesmosome–keratin on both alleles of either the LAMA3, the LAMB3, or the LAMC2 filament association in the two groups of laminin 5 or BP180 deficient genes, had similar morphometric measurements to the pyloric atresia nonlethal JEB patients were not significant (p Ͼ 0.05). Nevertheless associated JEB group, underscoring the likely role of laminin 5 in VOL. 110, NO. 2 FEBRUARY 1998 HEMIDESMOSOME–KERATIN FILAMENT INTERACTIONS 135

Figure 2. Transmission electron micro- scopy reveals altered connections between hemidesmosomes and keratin intermediate filaments in patients with different forms of JEB. Electron micrographs of skin from a normal control (a) and individuals with EB (b– i). Complete hemidesmosomes contain sub- basal dense plates (large arrowhead), inner plaques (small →), and outer plaques (large →) and show close association with bundles of keratin intermediate filaments (K) in normal skin (a). Herlitz JEB hemidesmosome plaques (large →) are reduced in size and sub-basal dense plates are absent. There is reduced association of keratin filaments with hemidesmosomes (b). The hemidesmosome plaque sizes are reduced and keratin filament association disrupted in the skin of patients with nonsense/nonsense BP180 mutations (d) and missense/nonsense mutation combinations (e), and there are less severe reductions in patients with laminin 5 defects (c). JEB associated with pyloric atresia exhibits a greater reduction in hemidesmosome–keratin filament association (f). Inner plaques are diminutive and sub-basal dense plates are miss- ing. The outer plaques are present (large →)in JEB with pyloric atresia (f). Autosomal recessive EB simplex associated with plectin defects shows the greatest reduction in hemidesmosome inner plaque size and in the number of hemidesmo- somes with keratin filament association (g). The hemidesmosome–keratin filament association is normal in skin from patients with dystrophic EB (h). A desmosome (D) between two basal keratinocytes in Herlitz JEB skin shows normal plaques with keratin filament association (i). Scale bar, 0.2 µm.

Table II. COL17A1 (BP180 gene) mutations and the hemidesmosome-anchoring filament complex. For example, lami- hemidesmosome assessment in non-lethal junctional nin 5 is a ligand for the α6β4 integrin (Niessen et al, 1994) and normal epidermolysis bullosa binding of this ligand with its integrin receptor may be important for subsequent intracellular signaling or maintenance of normal β4 integrin Casea Mutation Reference KIFb Inner attachment plaques function, including its direct or indirect interactions with basal cell keratins. 1 R1226X/41050insG McGrath et al, 1995 32.5% 32.5% During development of the human fetus, epidermal hemidesmosomes 2 3514ins25/G627V McGrath et al, 1996 78.9% 80.5% first appear concurrently with the earliest evidence of laminin 5 3 2518del10/? Unpublished 51.5% 58.1% expression at the dermal–epidermal junction, at a time also when α6β4 integrin can first be localized to hemidesmosomes.2 This, and other aCase 1 is shown in Fig 2d and case 2 is shown in Fig 2e. b work (Kurpakus et al, 1991), suggests that laminin 5 may have a critical Keratin intermediate filament role in the ordered assembly of hemidesmosome components. The role of the BP180 protein in the intracellular function of hemidesmosomes is hemidesmosome assembly and intracellular interactions. Some of these less easy to understand, largely because of the limited knowledge of interactions have been explored in greater depth by in vitro studies the intermolecular interactions involving this protein (Hopkinson showing that laminin 5 was capable of enhancing the keratin filament et al, 1995). association with the hemidesmosome, which was mediated by the The BP180 deficient nonlethal JEB patients in our study were integrin α6β4 and a plectin-like component (Baker et al, 1997). Similar, heterogeneous both phenotypically and genotypically. One individual but less severe changes in hemidesmosome numbers and keratin with two nonsense mutations showed severely disrupted hemidesmo- filament contacts were also evident in the groups of patients with some–keratin filament association, whereas skin from a patient with a nonlethal JEB, which included individuals with abnormalities of either nonsense/missense combination of BPAG2 mutations showed better laminin 5 or BP180. hemidesmosome–keratin filament association (Table II, Fig 2d vs 2e). Studies specifically examining the role of BP180 in hemidesmosome Hemidesmosome morphometry in this latter individual was similar to formation and function, particularly concerning the intracellular the laminin 5 deficient nonlethal JEB patients, two of whom had anchorage of the keratin filament network, appear to be limited. BP180 nonsense/missense combinations of mutations in the LAMB3 gene. is a transmembrane protein and the carboxyl terminal region of the The molecular pathology of this disorder may involve premature extracellular domain of the molecule has been localized to the zone termination codons on both alleles/inframe exons skipping or a bordering the lamina densa and lamina lucida (Bedane et al, 1997). nonsense/missense combination of mutations (McGrath et al, 1995a, Both the extracellular part of the BP180 molecule and laminin 5, 1996), and it is probable that the specific nature of these mutations in which is fully extracellular, are considered to be components of an individual patient has important direct consequences on hemidesmo- anchoring filaments (Rousselle et al, 1991; McGrath et al, 1994; Bedane some structure and keratin filament association. Ultrastructural findings et al, 1997) underlying the hemidesmosome plaques (Fig 2a). The apparent abnormalities of hemidesmosome linkage to keratin filaments associated with primary defects in laminin 5 or BP180 are likely to result from disruption or inadequacy of intermolecular interactions in 2McMillan JR, Br J Dermatol 134:589, 1996 (abstr.). 136 McMILLAN ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY in patients with JEB with pyloric atresia might, therefore, also show bullosa in association with aplasia cutis congenita and pyloric atresia. Acta Paediatr considerable heterogeneity reflecting the nature of the mutation. In Scand 71:155–160, 1982 Dowling J, Yu Q-C, Fuchs E: β4 integrin is required for hemidesmosome formation, cell this study, however, we have not examined the effect of specific adhesion and cell survival. J Cell Biol 134:559–572, 1996 mutations in laminin 5 or α6β4 genes on hemidesmosome structure Eady RAJ: Transmission electron microscopy. In: Skerrow D, Skerrow C (eds). Methods or intracellular connections. 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J Clin Invest 97:2289–2298, 1996 plasmic as well as the extracellular basement membrane connections Georges-Labouesse E, Messaddeq N, Yehia G, Cadalbert L, Dierich A, Le Meur M: Absence of integrin α6 leads to epidermolysis bullosa and neonatal death in mice. with hemidesmosomes, so the question remains why intracellular Nature Genet 13:370–373, 1996 disruption and blister formation are not seen. In fact they do occur, Guo L, Degenstein L, Dowling J, Yu Q-C, Wollmann R, Perman B, Fuchs E: Gene even though they may not be as pronounced as the extracellular targeting of BPAG1: Abnormalities in mechanical strength and cell migration in detachment. Intracellular cleavage has been reported in the skin of stratified epithelia and neurologic degeneration. Cell 81:233–243, 1995 Hashimoto I, Gedde Dahl T Jr, Schnyder UW, Anton Lamprecht I: Ultrastructural studies patients with JEB with pyloric atresia (Cowton et al, 1982; Smith, in epidermolysis bullosa hereditaria. IV. 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