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At least 19 recordsLinked to original sources

PubMed1993

Formation of hemidesmosomes in cells of a transformed murine mammary tumor cell line and mechanisms involved in adherence of these cells to laminin and kalinin.

Keratinocytes attach to an underlying basement membrane by adhesion junctions called hemidesmosomes. We have characterized a cell line, RAC-11P/SD, established from a murine mammary tumor, which differentiates into squamous epithelium and forms well defined hemidesmosomes. These hemidesmosomes contain the integrin alpha 6 beta 4 as well as the hemidesmosomal plaque proteins BP230 and HD1 and are associated with a matrix containing kalinin and laminin. We examined how these cells adhere to laminin and to kalinin present in matrices as well as immunopurified kalinin. We show that adhesion to laminin is energy dependent but does not require an intact actin-containing cytoskeleton. The affinity for kalinin proved to be greater and binding to kalinin was still observed when cells had been treated with deoxyglucose and azide to inhibit metabolic energy. Binding to laminin (or fragment E8), but not to kalinin was partially blocked by a monoclonal antibody specific for the integrin alpha 6 subunit, and only in the initial phase of adhesion. The antibody efficiently blocked adhesion to laminin of cells treated with the microfilament disrupting drug cytochalasin B, but only partially blocked the adhesion of cytochalasin B-treated cells to kalinin, while adherence of cells treated with deoxyglucose and azide to kalinin was blocked completely. The integrin alpha 6 beta 4 is redistributed to the basal surface during adhesion and then is organized into ring-like structures when cells are bound to laminin and localized into hemidesmosomes in cells adhered to kalinin. We suggest that anti-alpha 6 hinders the binding of the alpha 6 beta 4 integrins to its ligands laminin and kalinin, but cannot prevent adhesion when clustering of the integrin has become complete. In addition, there is evidence that adhesion to kalinin is mediated by a second receptor, which associates with the actin-containing cytoskeleton. The presence of such a second receptor is suggested because the cells can spread on kalinin, but not when they have been treated with cytochalasin B. On laminin spreading does not occur, irrespective of whether cells have been treated with cytochalasin B or not. The integrin alpha 3 beta 1, which has been identified as a receptor for kalinin but not for laminin, is strongly expressed in RAC-11P/SD cells and it seems likely that this integrin is responsible for spreading of cells on kalinin.

Animals↗

PubMed1993

Basement membrane proteins kalinin and nicein are structurally and immunologically identical.

BACKGROUND: The purpose of this investigation was 2-fold: (a) to compare two recently described proteins, the anchoring filament protein, kalinin and the hemidesmosome-associated protein, nicein (formerly called BM-600) which are both absent in junctional epidermolysis bullosa (JEB) Herlitz's disease; (b) to further define the structural defect in JEB Herlitz's disease. EXPERIMENTAL DESIGN: Cultured keratinocytes were analyzed with monoclonal antibodies (mAbs) against kalinin and nicein by indirect immunofluorescence. These mAbs were also used to immunoprecipitate radiolabeled proteins from keratinocyte cultures and to immunoaffinity purify proteins from keratinocyte conditioned culture medium. The precipitated or purified products were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, partial V8 protease digestion, and rotary shadowing. RESULTS: Kalinin and nicein mAbs show identical immunofluorescent staining patterns on cultured keratinocytes. Kalinin and nicein mAbs immunoprecipitate peptides from radiolabeled normal human keratinocyte cell and medium fractions that are electrophoretically identical. Partial V8 protease digestion patterns of the reduced 140 kilodalton peptides precipitated by nicein and kalinin mAbs are identical. Kalinin (like nicein) is absent from JEB Herlitz keratinocyte conditioned medium although K-laminin, another anchoring filament component, is present in these cultures. Kalinin, purified from conditioned keratinocyte medium by antibody affinity chromatography with K140-Sepharose (mAb against kalinin) and nicein purified from conditioned keratinocyte medium with GB3-Sepharose (mAb against nicein) are electrophoretically identical by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunologically identical by immunoblotting using kalinin and nicein mAbs. Rotary shadowed images of kalinin and nicein molecules are identical. CONCLUSIONS: We demonstrate that kalinin and nicein are identical by biochemical and immunologic analysis. We also verify that kalinin, like nicein, is absent in the conditioned medium of cultured JEB Herlitz keratinocytes, although another anchoring filament protein, K-laminin, is secreted by these cultures. These results correlate with previous immunofluorescent findings that show that while kalinin or nicein is absent in basement membranes of individuals with JEB Herlitz's disease, K-laminin appears to be present.

Antibodies, Monoclonal↗

PubMed1994

Kalinin is more efficient than laminin in promoting adhesion of primary keratinocytes and some other epithelial cells and has a different requirement for integrin receptors.

Kalinin was purified from squamous cell carcinoma (SCC25) spent culture media using an immunoaffinity column prepared from the mAb BM165. The affinity-purified material was separated by SDS-PAGE into three bands of 165-155, 140, and 105 kD identical to those obtained from normal human keratinocyte cultures and previously identified as kalinin. Kalinin promoted adhesion of a large number of normal cells and established cell lines with an activity similar to other adhesion molecules such as the laminin-nidogen complex, fibronectin, or collagen IV. However, kalinin was a much better substrate than laminin-nidogen complex for adhesion of cells of epithelial origin including primary human keratinocytes. Adhesion to kalinin was followed by cell shape changes ranging from rounded to fully spread cells depending on the cell types. The adhesion-promoting activity of kalinin was conformation dependent and was abolished by heat denaturation. mAb BM165 prevented cell adhesion to kalinin but not to other extracellular matrix substrates. However, either complete or partial inhibition was observed with different cells suggesting the existence of at least two cell-binding sites on the kalinin molecule. Experiments inhibiting cell adhesion with function-blocking anti-integrin subunit antibodies indicated that both alpha 3 beta 1 and alpha 6 beta 1 integrins are involved in the cellular interactions with kalinin, while for cell adhesion to classical mouse Engelbreth-Holm-Swarm laminin only alpha 6 beta 1 integrins, and not alpha 3 beta 1, appeared to be functional. Altogether, these results suggest that kalinin may fulfill additional functions than laminin, particularly for epithelial cells.

Animals↗

PubMed1994

The alpha 6 beta 4 integrin is a receptor for both laminin and kalinin.

Previously, we have establish K562 transfectants that express either alpha 6A beta 1 or alpha 6B beta 1 (K alpha 6A or K alpha 6B) on their surface. Both cell lines bind to laminin and kalinin after treatment with the beta 1-stimulatory antibody TS2/16. Here we introduce the full-length beta 4 cDNA into the alpha 6A- and alpha 6B-expressing K562 cells and selected stably transfected cells. The beta 4 subunit was expressed on the surface of both transfectants and it formed dimers with the alpha 6A or alpha 6B subunits. Immunoprecipitation and preclearing analyses revealed that both transfectants expressed alpha 6 beta 1, in addition to alpha 6 beta 4. While K alpha 6A and K alpha 6B cells required TS2/16 stimulation for binding to laminin or kalinin, adhesion of the unstimulated beta 4-transfected K alpha 6A and K alpha 6B cells to these matrix components was already substantial. This adhesion was mediated by both alpha 6 beta 1 and alpha 6 beta 4 since it was completely blocked by an alpha 6-specific antibody or by a combination of anti-beta 1 and anti-beta 4 antibodies, but only partially by either of these latter two antibodies alone. Adhesion to laminin was completely blocked by an antiserum to laminin fragment E8 as was the adhesion to kalinin by an antibody to kalinin, demonstrating the specificity of adhesion. Both transfectants always adhered more strongly to kalinin than to laminin. Furthermore, binding to kalinin was less well blocked by antibodies to beta 4 than binding to laminin, indicating that the affinity of alpha 6 beta 4 for kalinin is higher than that for laminin. The fact that alpha 6 beta 1 mediated adhesion without TS2/16 stimulation on the beta 4-transfected K alpha 6A and K alpha 6B cells suggests that some activation of alpha 6 beta 1 had occurred in these cells, even though binding was increased when they were actively stimulated by the antibody TS2/16. Finally, we show that Mn2+ induced binding of solubilized alpha 6 beta 4 to matrix containing kalinin, deposited by the murine cell line RAC-11P/SD. This binding was inhibited by the anti-alpha 6 mAb GoH3. Together, these results indicate that both alpha 6 beta 1 and alpha 6 beta 4 are receptors for laminin and kalinin and that there are no differences in ligand specificity between the A and B variants of the alpha 6 subunit when associated with either beta 1 or beta 4.

Animals↗

PubMed1992

Kalinin is abnormally expressed in epithelial basement membranes of Herlitz's junctional epidermolysis bullosa patients.

Kalinin is an extracellular adhesion molecule specific to epithelial basement membranes (BM) identified as a component of anchoring filaments of hemidesmosomes. This heterotrimeric protein is synthesized by cultured normal human keratinocytes and is involved in cell attachment. In indirect immunofluorescence studies, the epidermal BM of patients with junctional epidermolysis bullosa (JEB) of Herlitz's type were found not to be reactive with the anti-kalinin monoclonal antibodies ka146 and K140 and displayed a decreased immunoreactivity to two anti-kalinin antibodies cross-reacting with K-laminin, an anchoring filament component recently described. The intrinsic defect of JEB keratinocytes in the synthesis of kalinin was further documented by indirect immunofluorescence on in vitro cultures of these cells. In non-Herlitz JEB patients, staining of BM was constantly detected. Impairment of expression of kalinin correlated with the lack of reactivity to the monoclonal antibody GB3, which detects the BM component nicein/BM600. These results clearly demonstrate a defect of kalinin expression in epithelial basement membranes of Herlitz JEB patients and suggest that kalinin may play a role in the pathogenesis of the disease. Further studies are in progress to define possible relationships between kalinin and nicein.

Basement Membrane↗

PubMed1995

Mouse kalinin B1 (laminin beta 3 chain): cloning and tissue distribution.

BACKGROUND: Kalinin, an anchoring filament-associated protein, is a member of the laminin family which is believed to play a role in the attachment of particular epithelial cells to the basement membrane. EXPERIMENTAL DESIGN: A newborn mouse lung cDNA library was screened under low stringency conditions using as a probe a murine laminin beta 1 chain cDNA, an approach designed to identify novel laminin beta 1 chain homologs. RESULTS: We have identified a novel cDNA sequence comprising 4049 base pairs, with an open reading frame that potentially codes for 1168 amino acids, including a presumptive signal peptide. The gene was mapped to mouse chromosome 1. The amino acid sequence is homologous to laminin beta 1 and beta 2 chains, but is distinguished from these molecules by having a truncated short arm region. The sequence has considerable similarity to the human kalinin B1 chain (laminin beta 3), suggesting that it is murine beta 3 chain. Northern blot analysis demonstrated a single 4.0-kilobase species in the mouse newborn skin, lung, and Pam 212 cells. Immunolocalization studies using the 14-day embryo and adult mouse tissues demonstrated that the murine beta 3 chain is located at the dermal-epidermal junction of the skin and the basement membrane of the oral mucosa, oropharynx, and upper portions of the tracheobronchial tree. In situ hybridization studies of the mammary gland showed that the beta 3 mRNA chain was produced by the epithelial cells of the gland but not by the mesenchymal cells. Western blotting under nonreducing conditions using the antibody to beta 3 chain detected molecules of 400 and 440 kilodaltons (kDa) in the conditioned medium of human foreskin keratinocytes, A431 cells, and Pam 212 cells. Western blotting under reducing conditions detected only a single 140-kDa species, suggesting that beta 3 chain is present within disulfide-linked complexes. This 140-kDa species was contained in the kalinin complex immunoprecipitated by anti-kalinin B2 (laminin gamma 2) antibody from the conditioned medium of HT1080 cells, suggesting that both the beta 3 and gamma 2 chains are present in the multimeric complex. CONCLUSIONS: Murine laminin beta 3 chain shows considerable homology to the human homolog and has a tightly restricted tissue distribution, confined to the basement membrane of cells in contact with the external environment. In vitro studies indicate that the beta 3 chain participates in trimer formation with the gamma 2 chain and possibly with other laminin chains as well.

Amino Acid Sequence↗

PubMed1993

Labeling of fractured human skin with antibodies to BM 600/nicein, epiligrin, kalinin and other matrix components.

A variety of methods were used to fracture the dermal-epidermal junction (DEJ) of human skin. These included warm and hot phosphate buffered saline, trypsin, cold 1 M salt, potassium bromide and proteolytic digestion with dispase. The localization and sensitivity of basement membrane components (bullous pemphigoid antigen, BM 600/nicein, epiligrin, kalinin, laminin, collagens IV and VII (EBA antigen) and linkin) were determined after the DEJ was fractured by each method. We found that the basement membrane zone proteins, BM 600/nicein, epiligrin and kalinin remained with the dermal side of the DEJ fractured through the lamina lucida by cold salt, phosphate buffered saline and potassium bromide. BM 600/nicein, epiligrin and kalinin were not detected after treatment with trypsin. In contrast, laminin, another glycoprotein in the lamina lucida, was insensitive to all of the procedures, but co-localized to the dermal side of DEJ-fractured skin. We also found that separation of the DEJ with brief exposure of skin to 56 degrees C provided a useful substrate for testing the autoantibodies in the sera of patients with epidermolysis bullosa acquisita (EBA). Heat-separated skin can be prepared in a significantly shorter period of time than salt-separated skin.

Antibodies↗

PubMed1994

The mucin epiglycanin on TA3/Ha carcinoma cells prevents alpha 6 beta 4-mediated adhesion to laminin and kalinin and E-cadherin-mediated cell-cell interaction.

TA3/Ha murine mammary carcinoma cells grow in suspension, do not adhere to extracellular matrix molecules, but do adhere to hepatocytes and form liver metastases upon intraportal injection. Recently we showed that the integrin alpha 6 beta 4 on the TA3/Ha cells is involved in adhesion to hepatocytes. However, despite high cell surface levels of alpha 6 beta 4, TA3/Ha cells do not adhere to the alpha 6 beta 4 ligands laminin and kalinin. Here we show that this is due to the mucin epiglycanin that is highly expressed on TA3/Ha cells. Some monoclonal antibodies generated against epiglycanin induced capping of most of the epiglycanin molecules. TA3/Ha cells treated with these mAb did adhere to laminin and kalinin, and an epithelial monolayer was formed on kalinin, with alpha 6 beta 4 localized in HD1-containing hemidesmosome-like structures and E-cadherin at the cell-cell contact sites. Similar results were obtained after treatment of TA3/Ha cells with O-sialoglycoprotein endopeptidase which removes all epiglycanin. In addition, the enzyme induced E-cadherin-mediated cell-cell aggregation. Both treatments also enhanced the adhesion to hepatocytes, but given the potent antiadhesive effect of epiglycanin it is remarkable that nontreated TA3/Ha cells adhere to hepatocytes at all. We found that during this interaction, epiglycanin was redistributed. We conclude that epiglycanin can completely prevent both intercellular and matrix adhesion, but that this effect can be overcome in certain intercellular interactions because of the induced redistribution of the mucin.

Animals↗

PubMed1992

The anchoring filament protein kalinin is synthesized and secreted as a high molecular weight precursor.

Kalinin, a recently characterized novel protein component of anchoring filaments, has been shown to be involved in keratinocyte attachment to culture substrates and to dermis in vivo, and to exist in keratinocyte-conditioned culture medium in two heterotrimeric forms of 440 and 400 kDa (Rousselle, P., Lunstrum, G.P., Keene, D.R., and Burgeson, R.E. (1991) J. Cell Biol. 114, 567-576). This study demonstrates that kalinin is initially synthesized in a cell-associated form estimated to be 460 kDa. By second dimension reduced electrophoresis, V8 protease digestion, and immunoblot analysis, we demonstrate that the cell form contains nonidentical subunits of 200, 155, and 140 kDa. The 440-kDa medium form is derived from the cell form by extracellular processing of the 200-kDa subunit to 165 kDa, a step which also occurs in skin organ culture. The 400-kDa form is derived from the 440-kDa form by extracellular processing of the 155 kDa-subunit to 105 kDa. The cell form is secreted by keratinocytes, deposited onto culture substratum, and is the form which facilitates attachment and adhesion of growing and spreading keratinocytes. It is also the form initially synthesized in skin organ culture. Kalinin purified from tissue, which appears to facilitate epithelial-mesenchymal cohesion in vivo, is closely related to the 400-kDa medium form purified from culture.

Amnion↗

PubMed1993

The basement membrane protein BM-600/nicein codistributes with kalinin and the integrin alpha 6 beta 4 in human cultured keratinocytes.

The observation is reported that in low-passage human keratinocyte colonies cultured under conditions that allow full epidermal differentiation (i) the basement membrane protein BM-600/nicein, identified by the mAb GB3, is codistributed with laminin and collagen type IV as well as with the bullous pemphigoid antigen in footprints deposited by growing and migrating colonies; (ii) the integrin heterodimer alpha 6 beta 4 is codistributed with the same molecules suggesting its spatial association with basement membrane components; (iii) the distribution pattern of alpha 6 beta 4 and BM-600/nicein underneath individual cells is identical and is characterized by a typical "leopard skin" pattern complementary to the distribution of submembraneous F-actin microfilament network; (iv) a rabbit polyclonal antiserum to kalinin (R4012) used in double-label immunofluorescence staining with mAb GB3 shows that this protein has the same distribution as BM-600/nicein and this suggests that they are identically located; and (v) immunoprecipitation with mAb GB3 to BM-600/nicein and BM165 to kalinin shows identical bands suggesting that nicein and kalinin represent the same molecular entity. We suggest that alpha 6 beta 4 displays not only an adhesive role for keratinocytes in view of its reported association to hemidesmosomes but may also be involved in organizing the molecules of the epithelial extracellular matrix, including those forming the basement membrane zone and hemidesmosomes, a function proposed for other integrins in other cellular systems.

Actins↗

PubMedDate not supplied

Mesenchymal-epithelial interactions regulate gene expression of type VII collagen and kalinin in keratinocytes and dermal-epidermal junction formation in a skin equivalent model.

Anchoring fibrils constituted primarily of type VII collagen and anchoring filaments composed of kalinin are essential structural elements of the dermal-epidermal junction and critical for its stability. The role of fibroblasts in the production of these structural elements and the formation of the dermal-epidermal junction was studied by using a living skin equivalent model. This model had been modified such that keratinocytes and fibroblasts were allowed direct contact. After 2 weeks, immunohistochemical studies showed the linear deposition of type VII collagen and kalinin, as well as type IV collagen and alpha6 integrin at the dermal-epidermal junction. By electron microscopy, anchoring fibrils, a continuous lamina densa, and numerous hemidesmosomes were noted. Reverse transcriptase-polymerase chain reaction analysis showed an increased expression of both type VII collagen and kalinin genes in keratinocytes when they were in direct contact with fibroblasts. These results suggest that fibroblasts synthesize an extracellular matrix which favors keratinocyte adhesion and the formation of a dermal-epidermal junction by increasing the production and the further arrangement of dermal-epidermal junction components.

Journal Article↗

PubMed1995

Structural requirement for cell adhesion to kalinin (laminin-5).

Laminin-5 (kalinin) was purified from spent cell culture media (SCC25 cells) by affinity chromatography on monoclonal antibody BM165. The protein was recovered as a mixture of the typical polypeptides of 165-155, 140, and 105 kDa as judged b SDS-polyacrylamide gel electrophoresis analysis under reducing conditions. The amino acid composition of purified laminin-5 was in agreement with that compiled from the recently published cDNA sequences of the alpha 3-, beta 3-, and gamma 2-laminin chains. Moreover, the content of half-cystine residues in laminin-5 was about two-thirds that in laminin-1, which confirms the prediction of a smaller number of epidermal growth factor-like repeats in the amino-terminal portion of the three chains. The content of coiled-coil alpha-helices (27%) determined by CD spectroscopy was comparable to that reported for laminin-1, which indicates that the long arm portion of laminin-5 is equivalent to that of other laminin isoforms. The melting temperature was recorded at 72 degrees C by CD monitoring of unfolding and refolding of the coiled-coil structures during thermal denaturation and renaturation, respectively. The thermal stability of laminin-5 is therefore significantly higher than that of laminin-1 or alpha 2-chain-containing laminins, which suggests higher ionic interactions between the three polypeptide chains of laminin-5. Cell adhesion-promoting activity of laminin-5 was found to be strictly and entirely dependent on the presence of coiled-coil structures. It decreased gradually after heat denaturation of the protein above 65 degrees C and was totally abrogated at 75 degrees C. This is in contrast to laminin-1, which contains both conformation-dependent and -independent cell-binding sites on the long and short arm domains, respectively.

Binding Sites↗

PubMed1991

Kalinin: an epithelium-specific basement membrane adhesion molecule that is a component of anchoring filaments.

Basal keratinocytes attach to the underlying dermal stroma through an ultrastructurally unique and complex basement membrane zone. Electron-dense plaques along the basal surface plasma membrane, termed hemidesmosomes, appear to attach directly to the lamina densa of the basement membrane through fine strands, called anchoring filaments. The lamina densa is secured to the stroma through a complex of type VII collagen containing anchoring fibrils and anchoring plaques. We have identified what we believe is a novel antigen unique to this tissue region. The mAbs to this antigen localize to the anchoring filaments, just below the basal-dense plate of the hemidesmosomes. In cell culture, the antigen is deposited upon the culture substate by growing and migrating human keratinocytes. Addition of mAb to the cultures causes the cells to round and detach, but does not impair them metabolically. Skin fragments incubated with antibody extensively de-epithelialize. These findings strongly suggest that this antigen is intimately involved in attachment of keratinocytes to the basement membrane. This antigen was isolated from keratinocyte cultures by immunoaffinity chromatography. Two molecules are observed. The most intact species contains three nonidentical chains, 165, 155, and 140 kD linked by interchain disulfide bonds. The second and more abundant species contains the 165- and 140-kD chains, but the 155-kD chain has been proteolytically cleaved to 105 kD. Likewise, two rotary-shadowed images are observed. The larger of the two, presumably corresponding to the most intact form, appears as an asymmetric 107-nm-long rod, with a single globule at one end and two smaller globules at the other. The more abundant species, presumably the proteolytically cleaved form, lacks the distal small globule. We propose the name "kalinin" for this new molecule.

Cell Adhesion↗

PubMed1994

Molecular basis of the dystrophic and junctional forms of epidermolysis bullosa: mutations in the type VII collagen and kalinin (laminin 5) genes.

Epidermolysis bullosa (EB) is a group of heritable mechano-bullous skin diseases classified into three major categories, the simplex, junctional, and dystrophic forms, on the basis of the level of tissue separation within the dermal-epidermal basement membrane zone. Approaches of molecular biology have demonstrated that these three different forms of EB result from mutations in distinct genes: the simplex forms are due to mutations in the genes encoding keratins 5 and 14 expressed in basal keratinocytes; the junctional forms are associated with mutations in the kalinin/laminin 5 genes; and the dystrophic forms result from mutations in the type VII collagen gene (COL7A1). In this overview, we summarize our recent discoveries of pathogenic mutations in COL7A1, including premature termination codons that result in the severe, mutilating (Hallopeau-Siemens) type of recessive dystrophic EB and a glycine substitution in the collagenous region resulting in dominant dystrophic EB. Furthermore, we present evidence that implicates mutations in the kalinin/laminin 5 gamma 2 chain gene (LAMC2) in some forms of junctional EB. This information has provided the basis for DNA-based prenatal diagnosis during the first trimester of gestation, and sets the stage for the application of gene therapy to these devastating skin diseases in the future.

Cell Adhesion Molecules↗

PubMed1952