Hepatic stellate cells express synemin, a protein bridging intermediate filaments to focal adhesions.

BACKGROUND AND AIMS: In the liver, stellate cells play several important (patho)physiological roles. They express a broad but variable spectrum of intermediate filament (IF) proteins. The aim of this study was to investigate the expression and functions of the intermediate filament protein synemin in hepatic stellate cells (HSCs). METHODS: In isolated and cultured rat HSCs, synemin expression was examined by quantitative reverse transcriptase polymerase chain reaction, western blotting, and immunocytochemistry. Protein-protein interaction between synemin and possible binding partners was investigated by co-immunoprecipitation and confocal microscopy. RESULTS: Expression of synemin was significantly downregulated with increased culture time. In 1-day cultured HSCs, synemin associated with other IF proteins (GFAP, desmin, and vimentin), and with the focal adhesion proteins vinculin and talin, but not with alpha-actinin or paxillin. Synemin IF and focal adhesion proteins co-localised in long slender processes, but not in the lamellipodia. In human and rat liver tissue, the presence of synemin was investigated by immunohistochemistry. In normal rat and human livers, synemin immunoreactivity was found in HSCs, smooth muscle cells of hepatic arterioles, and nerve bundles in portal tracts, but not in portal fibroblasts. In CCl4-intoxicated rat livers and in human cirrhotic livers, immunoreactivity for synemin in the parenchymal tissue was decreased. Thus synemin was expressed in quiescent HSCs but not in portal fibroblasts; and synemin expression decreased with HSC activation in vivo during chronic liver damage and with HSC activation in culture. CONCLUSIONS: Synemin forms heteropolymeric filaments with type-III IF proteins and acts as a bridging protein between IFs and a specific type of focal adhesions.

Transient translocation of hemidesmosomal bullous pemphigoid antigen 1 from cytosol to membrane fractions by 12-O-tetradecanoylphorbol-13-acetate treatment and Ca2+-switch in a human carcinoma cell line.

We previously showed that 12-O-tetradecanoylphorbol-13-acetate (TPA) and Ca2+-switch from low (0.07 mM) to normal (1.87 mM) concentration in culture medium, which were also linked to activation of protein kinase C (PKC), lead to phosphorylation of 180 kDa-bullous pemphigoid antigen (BPAG) 2, but not of 230 kDa-BPAG1, and possibly to its disassembly from hemidesmosomes in a human squamous cell carcinoma cell line (DJM-1). In this study, we examined the effects of TPA and Ca2+-switch on intracellular localization of BPAG1 by immuno-blotting and immuno-fluorescence microscopy with monoclonal antibodies to the antigen after sub-cellular fractionation. In DJM-1 cells cultured in low Ca2+ medium, BPAG1 was detected as phosphate buffered saline-soluble (cytosolic), Triton X-100 soluble (roughly membrane-associated) and Triton X-100 insoluble (cytoskeleton-bound) forms, whereas in normal Ca2+-grown cells only as cytosolic and cytoskeleton-bound forms. In normal Ca2+-cultured cells, TPA (50 nM) caused a complete translocation of BPAG1 from cytosol to membrane fractions within 10 min, that was inhibited by pretreatment with H7 (a selective PKC inhibitor) at 40 microM. After 30 min and 4 h of TPA-treatment, BPAG1 was exclusively detected in cytoskeleton fractions. Morphologically, immuno-fluorescence microscopy showed that treatment caused a marked reduction of BPAG1 from the cytoplasm and generated a linear pattern at cell-cell contacts, suggesting translocation of BPAG1 from the cytosol to the plasma membrane. In contrast, the Ca2+-switch from low to normal caused a prominent increase of BPAG1, both in cytosolic and membrane-associated forms after 4 h, that was inhibited both with H7 and cycloheximide (an inhibitor of protein synthesis) at 70 microM, suggesting a role for PKC and BPAG1 synthesis in these Ca2+-induced effects. These results suggest that TPA and Ca2+-switch induced BPAG1 translocation to membrane fractions possibly mediated by PKC-activation. Furthermore, whereas TPA affects the redistribution of BPAG1 among their pools without inducing their synthesis, Ca2+-switch induces both membrane translocation and synthesis of BPAG1, suggesting involvement of signaling other than PKC pathways in control of BPAG1 synthesis.

Cicatricial pemphigoid with circulating autoantibodies to beta4 integrin, bullous pemphigoid 180 and bullous pemphigoid 230.

Cicatricial pemphigoid is a heterogeneous group of autoimmune subepidermal blistering diseases associated most commonly with autoantibodies to bullous pemphigoid (BP)180 and less frequently with those to laminin 5 or type VII collagen. In addition, a few cases have been described with autoantibodies to the beta4 subunit of alpha6beta4 integrin. We describe a patient with extensive disease of ocular, oral, pharyngeal, laryngeal and genital mucous membranes that healed with scarring of conjunctivae. IgG autoantibodies bound to the dermal-epidermal junction on direct immunofluorescence (IF) microscopy and to the epidermal side of 1 mol L(-1) NaCl-split skin on indirect IF microscopy. Our patient's circulating IgG recognized a 205-kDa protein in extracts of 293T cells transfected with the beta4 subunit of alpha6beta4 integrin and in the cell extract of DJM-1 cells. Our patient's IgG and IgA autoantibodies also reacted with full-length BP180 derived from epidermal extracts and the ectodomain of BP180 (LAD-1) derived from culture supernatant of keratinocytes. In addition, a weak IgG reaction with BP230 was noted. The disease rapidly responded to dexamethasone-cyclophosphamide pulse therapy, and immunoblot reactivity to both beta4 integrin and BP180 decreased according to disease activity.

Identification of the hemidesmosomal 500 kDa protein (HD1) as plectin.

HD1 is a 500 kDa hemidesmosomal plaque protein recognized by monoclonal antibody mAb-121. Recent research on inherited skin disease has suggested that it might be identical to plectin or an isoform. To cast light on this question, we have prepared several monoclonal antibodies that recognize a 500 kDa protein in the hemidesmosome fraction. Unexpectedly, some staining pattern heterogeneity was observed on immunofluorescence microscopy. Attention was focused on two monoclonal antibodies which gave different localization in bovine skin and retinal pigment epithelial cells. Determination of the amino-terminal sequence of an antigenic 100 kDa polypeptide fragment derived from the 500 kDa component of an insoluble fraction of bovine hepatocytes revealed it was identical to that of plectin. Using the two antibodies, we screened a cDNA library derived from BMGE+H, a bovine mammary gland epithelial cell line. The isolated cDNA clones corresponded to the rod domain of bovine plectin, with two separate epitope regions for each of the antibodies. From these results we conclude that the hemidesmosomal 500 kDa component HD1 is identical to plectin. As judged on rough estimation of molar ratios on this basis, hemidesmosomes are composed of plectin, BP230, the integrin beta4 subunit, and alpha6 in a 1:1:1:1 ratio.

Hemidesmosomes and their unique transmembrane protein BP180.

Hemidesmosomes are adhesion complexes responsible for linking keratin intermediate filaments of stratified and complex epithelia to components of the extracellular matrix such as collagen fibrils. Over the past several years, it has become clear that there are at least five hemidesmosomal proteins, including HD1/plectin and BP230 as cytoplasmic plaque proteins and integrin alpha6beta4 and BP180 as transmembrane proteins. Among them, BP180 is unique as a transmembrane protein because of its collagenous extracellular domain. Recent biochemical and ultrastructural analyses have revealed its molecular configuration and nature as a major component of anchoring filaments connecting hemidesmosomes to the basement membrane. These results indicate that BP180 is a new type of adhesion receptor. In addition to biochemical analyses of these hemidesmosomal proteins, recent studies on patients with inherited skin blistering diseases and on knockout mice have demonstrated roles in hemidesmosome formation and stabilization, as well as unexpected, novel functions.

Cleavage of BP180, a 180-kDa bullous pemphigoid antigen, yields a 120-kDa collagenous extracellular polypeptide.

The hemidesmosome (HD) is a cell-to-substrate adhesion apparatus found in stratified and complex epithelia. One of the putative cell-matrix adhesion molecules present in the HD is the 180-kDa bullous pemphigoid antigen (BP180), also termed type XVII collagen. In our previous study, using a monoclonal antibody (mAb) 1337, we have detected a 120-kDa collagenase-sensitive polypeptide in the HD fraction (Uematsu, J. and Owaribe, K. (1993) Cell Struct. Funct. 18, 588 (abstr.)). The present study was undertaken to assess the relation of the 120-kDa polypeptide to this BP180. Immunofluorescence microscopy of bovine skin revealed the basement membrane zone of skin to be stained clearly with mAb 1337, whereas the lateral surfaces of basal cells, which were decorated by typical antibodies against BP180, were not. The antibody did not detect HDs in cultured cells but rather in the culture medium. These results indicate a localization of mAb 1337 antigen distinct from BP180. However, the same polypeptide was also recognized by monoclonal antibodies to the extracellular but not the cytoplasmic part of BP180, and found to react with a polyclonal antibody against the non-collagenous 16A domain of BP180. Therefore, the polypeptide was identified as an extracellular fragment of BP180. mAb 1337 immunoprecipitated the 120-kDa fragment from the medium, but not the 180-kDa molecule of BP180 extracted from cultured cells, indicating that the antibody specifically recognizes the fragment. The mAb 1337 apparently recognizes a unique epitope that is exposed or formed by the cleavage. Hence, the staining pattern observed for bovine skin demonstrated the presence of the 120-kDa extracellular fragment. Rotary shadow electron microscopy of affinity-purified 120-kDa fragments demonstrated that they have the unique molecular shape consisting of a central rod and a flexible tail, without the globular head that is present in the BP180 molecule. From these results, we conclude that mAb 1337 shows unique epitope specificity, recognizing only the 120-kDa extracellular fragment of BP180, which is constitutively cleaved on the cell surface as a 120-kDa fragment both in in vivo and in vitro.

Internalization of the 180 kDa bullous pemphigoid antigen as immune complexes in basal keratinocytes: an important early event in blister formation in bullous pemphigoid.

We have previously shown that the 180 kDa bullous pemphigoid antigen (BPAG2) is distributed on the lateral-apical (as a pool) and ventral (as hemidesmosomes) cell membranes of monolayer cultured keratinocytes and that addition of IgG purified from bullous pemphigoid (BP) patients (BP-IgG) causes the internalization of immune complexes of BPAG2 and BP-IgG from the lateral-apical cell membrane. This internalization of BPAG2 is believed to inhibit the Ca2+ induced reformation of hemidesmosomes on the ventral cell membrane, possibly by inhibiting the supply of the antigen from the lateral-apical to the ventral membranes to form hemidesmosomes. The purpose of this paper is to examine, by using biopsy specimens from BP patients (12 cases), whether or not this internalization of BPAG2 is generated in situ. The fates of BPAG2, 230 kDa BPA (BPAG1) and bound BP-IgG were traced by immunofluorescence microscopy using monoclonal antibodies to BPAG1, BPAG2 and human IgG. In more than half of the lesional and perilesional biopsy specimens, internalization of BPAG2 into the basal cells was observed, while in normal skin BPAG2 was detected on the whole surface of the basal cells without its internalization. No internalization of BPAG1 was detected in BP and normal epidermis. These results suggest that binding of BP-IgG on the lateral-apical cell surface of basal cells causes internalization of BPAG2 in situ in the epidermis of BP patients similar to that seen in cultured cell systems, and that this internalization of immune complexes of BPAG2 and BP-IgG may play an important part in blister formation in BP.

Demonstration of the molecular shape of BP180, a 180-kDa bullous pemphigoid antigen and its potential for trimer formation.

The 180-kDa bullous pemphigoid antigen (BP180) is a hemidesmosomal transmembrane glycoprotein comprising interrupted collagen domains in its extracellular part. BP180 is also termed type XVII collagen. But the question of whether it actually takes a collagen-like triple helical conformation in vivo has remained unanswered. Using a monoclonal antibody, we found that a subpopulation of BP180 localizes at the lateral surfaces of corneal basal cells and cultured cells, in addition to the basal surface. This subpopulation of BP180 could be solubilized by 0.5% Triton X-100 and, among examined cell lines, was found to be most abundant in BMGE+H, a bovine mammary gland epithelial cell line. The Triton-soluble fraction of BMGE+H cells was used for characterization. On sucrose gradient centrifugation, the soluble BP180 demonstrated a value of approximately 7 S, and chemical cross-linking experiments revealed a trimer form. The calculated frictional ratio, f/f0 = 2.8, suggests an asymmetric configuration. For further characterization, we purified the soluble BP180 by immunoaffinity column chromatography using an anti-BP180 monoclonal antibody. Rotary shadowing images of the purified BP180 showed a quaver-like molecule consisting of a globular head, a central rod, and a flexible tail. With regard to the primary structure and species comparisons, the central rod, 60-70 nm in length, probably corresponds to the largest collagenous region, forming a collagen-like triple helix, in human form. The globular head and the flexible tail seem to correspond to the cytoplasmic and the interrupted collagenous region, respectively, of the extracellular portions. In conclusion, the present demonstration of the entire configuration of BP180, with a collagen-like trimer in its extracellular part, suggests that BP180 is one of the major components of anchoring filaments.

A hemidesmosomal transmembrane collagenous molecule, the 180-kDa bullous pemphigoid antigen (BPA II), is phosphorylated with 12-O-tetradecanoylphorbol-13-acetate in a human squamous cell carcinoma cell line (DJM-1).

We have previously shown that the 180-kD bullous pemphigoid antigen (BPAII), which is a transmembrane collagenous protein of hemidesmosomes, is distributed at adhesion sites on glass coverslips on the basal membrane forming a concentric ring, or arch pattern, in a human squamous cell carcinoma cell line (DJM-1), when studied by immunofluorescence microscopy using monoclonal antibodies to BPA II. This concentric ring/arch pattern of "footsteps" of BPA II has been shown to be collapsed in association with a transient activation of protein kinase C by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). In the present study, therefore, the effects of TPA on the phosphorylation of BPA II was examined. DJM-1 cells, which were metabolically labelled with [32Pi], were lysed and the extracts were subjected to immunoprecipitation with anti-BPAII and anti-230 kDa bullous pemphigoid antigen (BPAI) monoclonal antibodies. The results showed that only BPA II, but not BPA I, was phosphorylated at serine residues before TPA treatment. After TPA treatment phosphorylation was prominently increased so as to generate a 190 kDa-phosphorylated peptide. This 190-kDa peptide was reacted with anti-BPA II monoclonal antibodies by immunoblotting, and it was not detected when cells were pretreated with a specific protein kinase C inhibitor (H7) before TPA treatment, suggesting that the 190 kDa peptide is phosphorylated BPAII with TPA. Prolonged treatment with TPA abolished both of 180- and 190-kDa BPA II from Triton X-100-soluble fractions. These findings suggest that the BPA II, but not BPA I, is a substrate of protein kinase C, and the generation of 190-kDa-phosphorylated BPA II has a key role in the TPA-induced collapse of the assembly of BPA II on the basal plasma membrane, probably, at hemidesmosomes.

Antibody-binding to the 180-kD bullous pemphigoid antigens at the lateral cell surface causes their internalization and inhibits their assembly at the basal cell surface in cultured keratinocytes.

We demonstrated the effects of monoclonal antibodies to the 180-kD and 230-kD BP antigens (BPA) and of BP sera on Ca(++)-induced formation of hemidesmosomes in cultured human keratinocytes (a cell line, DJM-1) by immunofluorescence microscopy. Under low Ca++ (0.07 mM) conditions, the 180-kD and 230-kD BPAs were distributed homogeneously on the basal plasma membrane, while they formed a peculiar concentric ring or arch (ring/arch) arrangement in high-Ca++ (1.87 mM) medium. On the other hand, the apical-lateral cell membrane was stained homogeneously with antibodies to the 180-kD BPA, but not to the 230-kD BPA, both in low and high Ca++ media. The low-high Ca++ switch at first caused disappearance of the antigen from the basal plasma membrane and then formed the high-Ca++ ring/arch pattern within 3 hrs. In this system, monoclonal antibodies to the 180-kD and 230-kD BPAs and the sera from 5 BP patients, 2 pemphigus vulgaris (PV) patients, and 4 normal volunteers were added into the culture media. The addition of anti-180-kD BPA antibodies or any BP serum caused the internalization of the 180-kD BPA from the apical-lateral cell membrane and inhibited the Ca(++)-induced formation of the ring/arch pattern on the basal membrane, possibly by inhibiting the movement of the antigen from the lateral to the basal membrane to form hemidesmosomes.(ABSTRACT TRUNCATED AT 250 WORDS)

A possible cell-biologic mechanism involved in blister formation of bullous pemphigoid: anti-180-kD BPA antibody is an initiator.

In this short review, we summarize the results of our recent studies on the effects of anti-bullous-pemphigoid antigen (BPA) antibodies and BP sera on the hemidesmosome in cultured keratinocytes (DJM-1 cells) as examined by immunofluorescence microscopy. The 180-kD and the 230-kD BPAs localized on the basal plasma membrane showed a homogeneously dotted pattern in cells grown with low Ca2+ (0.07 mM), while they formed a peculiar concentric ring or arch (ring/arch) pattern in cells grown with high Ca2+ (1.87 mM). In addition, the 180-kD BPA was distributed also on the lateral/apical cell membrane, and the 230-kD BPA was found in the cytoplasm. The high Ca2+ ring/arch arrangement of BPAs was formed within 3 h after the low-high Ca2+ switch. Anti-180-kD BPA monoclonal antibodies (MAbs) and BP sera, but not anti-230-kD BPA MAbs, which were added into this system, caused the internalization of the 180-kD BPA from the lateral/apical cell membrane and inhibited the formation of the ring/arch pattern. These results suggest that autoantibodies to the 180-kD, but not to the 230-kD, BPAs may directly bind to the antigen on the cell surface of the basal cells and disturb the formation of hemidesmosomes. The 180-kD BPA appears to be an initiator of blister formation.