The epithelium-tooth interface--a basal lamina rich in laminin-5 and lacking other known laminin isoforms

The attachment of the marginal gingiva to the tooth surface is mediated by a thin nonkeratinized epithelium termed the junctional epithelium (JE). Ultrastructural studies have revealed that the attachment of the JE to the tooth surface occurs through hemidesmosomes (HD) and a basal lamina-like extracellular matrix termed the internal basal lamina (IBL). We have previously shown that neither type IV collagen nor prototypic laminin, two common components of basement membranes (BM), is present in the IBL between the epithelium and the tooth. In the present study, we show that laminin-5 is a major component of the IBL in both rodent and human tissues. By using in situ hybridization, we also show that the cells of the JE express the LAMC2 gene of laminin-5. In other parts of gingival epithelium, LAMC2 gene expression is less prominent. Our results indicate that the epithelium-tooth interface is a unique structure wherein epithelial cells are induced to secrete a basal lamina containing laminin-5 and no other presently known laminin isoform.     

The effects on inhibition of corneal neovascularization after human amniotic membrane transplantation in severely damaged rabbit corneas

Human amniotic membrane isolated from the placenta contained basement membrane components such as type IV collagen, laminin, and 6 and 4 integrins, all of which remained detectable while preserved in glycerin for one week. One month after the n-heptanol removal of the total corneal epithelium and the limbal lamellar keratectomy, all rabbit eyes carried features of limbal deficiency, including conjunctival epithelial ingrowth, vascularization and chronic inflammation. Ten control eyes then received a total keratectomy, and 13 experimental eyes received an additional amniotic membrane transplantation. Three-month follow-ups revealed that all control corneas were revascularized to the center with granuloma and retained a conjunctival phenotype. In contrast, in the experimental groups, 5 corneas became clear with either minimal or no vascularization; the rest had either mild peripheral (5) or total (3) vascularization and more cloudy stroma. Using monoclonal antibodies for epithelial markers and matrix components, we concluded that the success correlated with the return of a cornea-like epithelial phenotype and the preservation of the amniotic membrane, whereas the failure maintained a conjunctival epithelial phenotype and the amniotic membrane was either partially degraded or covered by host fibrovascular stroma. Measures taken to facilitate the former might prove this procedure clinically useful for ocular surface reconstruction.     

alpha3beta1 Integrin is required for normal development of the epidermal basement membrane

Integrins alpha3beta1 and alpha6beta4 are abundant receptors on keratinocytes for laminin-5, a major component of the basement membrane between the epidermis and the dermis in skin. These integrins are recruited to distinct adhesion structures within keratinocytes; alpha6beta4 is present in hemidesmosomes, while alpha3beta1 is recruited into focal contacts in cultured cells. To determine whether differences in localization reflect distinct functions of these integrins in the epidermis, we studied skin development in alpha3beta1-deficient mice. Examination of extracellular matrix by immunofluorescence microscopy and electron microscopy revealed regions of disorganized basement membrane in alpha3beta1-deficient skin. Disorganized matrix was first detected by day 15.5 of embryonic development and became progressively more extensive as development proceeded. In neonatal skin, matrix disorganization was frequently accompanied by blistering at the dermal-epidermal junction. Laminin-5 and other matrix proteins remained associated with both the dermal and epidermal sides of blisters, suggesting rupture of the basement membrane itself, rather than detachment of the epidermis from the basement membrane as occurs in some blistering disorders such as epidermolysis bullosa. Consistent with this notion, primary keratinocytes from alpha3beta1-deficient skin adhered to laminin-5 through alpha6 integrins. However, alpha3beta1-deficient keratinocytes spread poorly compared with wild-type cells on laminin-5, demonstrating a postattachment requirement for alpha3beta1 and indicating distinct roles for alpha3beta1 and alpha6beta4. Our findings support a novel role for alpha3beta1 in establishment and/or maintenance of basement membrane integrity, while alpha6beta4 is required for stable adhesion of the epidermis to the basement membrane through hemidesmosomes.     

Characterization of dp6troglycan-laminin interaction in peripheral nerve

Dystoroglycan is encoded by a single gene and cleaved into two proteins, alpha and beta-dystroglycan, by posttranslational processing. The 120kDa peripheral nerve isoform of alpha-dystroglycan binds laminin-2 comprised of the alpha 2, beta 1, and gamma 1 chains. In congenital muscular dystrophy and dy mice deficient in laminin alpha 2 chain, peripheral myelination is disturbed, suggesting a role for the dystroglycan- laminin interaction in peripheral myelinogenesis. To begin to test this hypothesis, we have characterized the dystroglycan-laminin interaction in peripheral nerve. We demonstrate that (1) alpha-dystroglycan is an extracellular peripheral membrane glycoprotein that links beta-dystroglycan in the Schwann cell outer membrane with laminin-2 in the endoneurial basal lamina, and (2) dystrophin homologues Dp116 and utrophin are cytoskeletal proteins of the Schwann cell cytoplasm. We also present data that suggest a role for glycosylation of alpha-dystroglycan in the interaction with laminin.     

Intestinal epithelial restitution. Involvement of specific laminin isoforms and integrin laminin receptors in wound closure of a transformed model epithelium

Disruptions in the mucosal lining of the gastrointestinal tract reseal by epithelial cell migration, a process termed restitution. We examined the involvement of laminin isoforms and their integrin receptors in restitution using the intestinal epithelial cell line T84. T84 cells express primarily laminins 5, 6, and 7 as indicated by immunostaining using laminin subunit-specific monoclonal antibodies (MAbs). A MAb (BM2) specific for the laminin alpha 3 subunit, a component of laminins 5, 6, and 7, completely inhibited the closure of mechanical wounds in T84 monolayers. Confocal microscopy using MAbs BM2 (laminin alpha 3 subunit) and 6F12 (laminin beta 3 subunit) revealed that laminin-5 is deposited in a basal matrix that extends into the wound. The MAbs 4E10 (laminin beta 1 subunit) and C4 (laminin beta 2 subunit) stained the lateral membranes between T84 cells. This staining was enhanced in cells adjoining wounds. Because T84 cells stained faintly with MAbs 4C7 (laminin alpha 1 subunit) and with MAbs 4F11 and 1B4 (laminin alpha 2 subunit), we suggest that expression of laminins 6 and 7 is enhanced in response to wounding. The alpha 3 beta 1 integrin and the alpha 6-containing integrins function in wound closure because MAbs specific for the beta 1 integrin subunit (MAb13), the alpha 3 subunit (IVA5), and the alpha 6 subunit (2B7) potently inhibited T84 migration into wounds. Immunofluorescence using UMA9, a beta 4-integrin-specific MAb, revealed that alpha 6 beta 4 integrin exists in a Triton-X-100-insoluble structure at the basal surface and that the staining of this structure is enhanced in cells adjoining wounds. In addition, a Triton-X-100-soluble pool of alpha 6 beta 4, as well as alpha 3 beta 1 and presumably alpha 6 beta 1, was found along lateral surfaces of T84 cells. On flattened cells adjoining wounds, staining for these integrins was distributed diffusely, suggesting a redistribution that accompanies cell migration. Taken together, these data suggest that wound-induced epithelial cell migration is a finely tuned process that is dependent upon the regulated function and localization of specific laminins and their integrin receptors.     

Expression of laminin alpha 1, alpha 5 and beta 2 chains during embryogenesis of the kidney and vasculature

Laminins, found predominantly in basement membranes, are large glycoproteins consisting of different subsets of alpha, beta and gamma chain subunits. To resolve conflicting data in the literature concerning coexpression of alpha 1 and beta 2 chains, expression of alpha 1 chain was studied with two different antisera against the E3 fragment of laminin alpha 1 chain. Expression of the alpha 1 chain was seen in several types of epithelial basement membranes throughout development, but its expression in rat glomerular basement membranes and some other types of epithelial basement membranes occurred only during early stages of development. By contrast, beta 2 chains were detected by immunofluorescence only during advanced stages of glomerulogenesis and vascular development. By Northern and Western blots, beta 2 chains were detected somewhat earlier, but in situ hybridization revealed that beta 2 chain was also confined to vasculature during the earlier stages. It thus seems that, in the tissues studied here, the expression of alpha 1 and beta 2 chains was mutually exclusive. To explore whether the newly described alpha 5 chain is expressed in locations lacking alpha 1 chain, expression of alpha 5 chain was studied by Northern blots and in situ hybridization. The alpha 5 chain was not uniformly expressed in all embryonic epithelial cell types but was present mainly in epithelial sheets which produce very little alpha 1 chain. There also appeared to be a developmental trend, with alpha 1 chain appearing early and alpha 5 later, in maturing epithelial sheets. The alpha 5 chain could be a major alpha chain of the adult glomerular basement membrane.     

Optimal versus maximal safety of the blood transfusion chain in The Netherlands; results of a conference. College for Blood Transfusion of the Dutch Red Cross

The distribution of major components of the basement membrane, such as type IV collagen, laminin, and heparan sulfate proteoglycan (HSPG), was investigated in the rat cochlear duct. Immunofluorescence demonstrated that type IV collagen, laminin and HSPG were distributed along capillaries in the cochlear duct, including the stria vascularis, spiral ligament, spiral prominence and spiral limbus. Additionally, type IV collagen, laminin and HSPG were found to be distributed from the basement membrane of Reissner's membrane to that of the spiral prominence in a linear pattern. The scala media was surrounded by these basement membrane components, demarcating endolymph from perilymph, along epithelial cells except at the stria vascularis. These findings suggest that type IV collagen, laminin and HSPG create the anatomical separation between endolymph and perilymph, thus indicating that they may be involved in the regulation of fluid transport between the endolymph and perilymph.     

Alterations of corneal extracellular matrix after multiple refractive procedures: a clinical and immunohistochemical study

PURPOSE: To describe the clinical course and alterations of the corneal extracellular matrix (ECM) and basement membrane (BM) in a cornea after hexagonal keratotomy, transverse keratotomies, and keratomileusis. METHODS: Frozen sections of this cornea and of 12 normal corneas were studied by immunofluorescence with specific antibodies. The patient history was analyzed to allow a clinical correlation. RESULTS: In the treated cornea, keratotomy scars and subepithelial fibrosis with neovascularization were seen. Around and beneath the epithelial plugs and along the keratotomy scars, deposits of types III, VI, VIII, and XIV collagen; fibrillin-1; fibronectin; and tenascin-C were found, together with short streaks of types IV (alpha 1-alpha 2) and VII collagen, laminin-1 and -5, entactin, and perlecan. alpha 3-alpha 4 Type IV collagen chains were abnormally absent from the BM around the epithelial plugs. At the edges of the keratomileusis flap, subepithelial fibrosis areas were found, with abnormal deposits of eight different collagen types, perlecan, fibronectin, fibrillin-1, and tenascin-C. The major part of the flap interface did not show ECM abnormalities. ECM alterations outside the scarred areas included the appearance of tenascin-C in the stroma and of alpha 1-alpha 2 type IV collagen in the epithelial BM, and the disappearance of fibronectin from Descemet's membrane. CONCLUSION: Five years after surgery, the treated cornea still presented BM abnormalities at sites of keratotomy scars and epithelial plugs. Several ECM components were abnormally expressed outside the scarred areas, consistent with an ongoing fibrosis in the treated cornea.     

Multi-component T1 relaxation and magnetisation transfer in peripheral nerve

Corneas of diabetic patients have abnormal healing and epithelial adhesion, which may be due to alterations of the corneal extracellular matrix (ECM) and basement membrane (BM). To identify such alterations, various ECM and BM components and integrin receptors were studied by immunofluorescence on sections of normal and diabetic human corneas. Age-matched corneas from 15 normal subjects, 10 diabetics without diabetic retinopathy (DR), and 12 diabetics with DR were used. In DR corneas, the composition of the central epithelial BM was markedly altered, compared to normal or non-DR diabetic corneas. In most cases the staining for entactin/nidogen and for chains of laminin-1 (alpha1beta1gamma1) and laminin-10 (alpha5beta1gamma1 was very weak, discontinuous, or absent over large areas. Other BM components displayed less frequent changes. The staining for alpha3beta1 (VLA-3) laminin binding integrin was also weak and discontinuous in DR corneal epithelium. Components of stromal ECM remained unchanged even in DR corneas. Therefore, distinct changes were identified in the composition of the epithelial BM in DR corneas. They may be due to increased degradation or decreased synthesis of BM components and related integrins. These alterations may directly contribute to the epithelial adhesion and wound healing abnormalities found in diabetic corneas.     

Role of laminin polymerization at the epithelial mesenchymal interface in bronchial myogenesis

Undifferentiated mesenchymal cells were isolated from mouse embryonic lungs and plated at subconfluent and confluent densities. During the first 5 hours in culture, all the cells were negative for smooth muscle markers. After 24 hours in culture, the mesenchymal cells that spread synthesized smooth muscle alpha-actin, muscle myosin, desmin and SM22 in levels comparable to those of mature smooth muscle. The cells that did not spread remained negative for smooth muscle markers. SM differentiation was independent of cell-cell contact or proliferation. In additional studies, undifferentiated lung mesenchymal cells were cocultured with lung embryonic epithelial cells at high density. The epithelial cells aggregated into cysts surrounded by mesenchymal cells and a basement membrane was formed between the two cell types. In these cocultures, the mesenchymal cells in contact with the basement membrane spread and differentiated into smooth muscle. The rest of the mesenchymal cells remained round and negative for smooth muscle markers. Inhibition of laminin polymerization by an antibody to the globular regions of laminin beta1/gamma1 chains blocked basement membrane assembly, mesenchymal cell spreading and smooth muscle differentiation. These studies indicated that lung embryonic mesenchymal cells have the potential to differentiate into smooth muscle and the process is triggered by their spreading along the airway basement membrane.     

Secular trend in body height since the Neolithic period

Rat mesangial cells express two unique isoforms of laminin which can be modulated by culture medium composition. To define further the nature of laminin expressed by cultured rat mesangial cells, synthesis of individual laminin chains, as well as their trimeric association, was examined. Based on data from Northern analysis of mRNA expression, immunoblots, immunofluorescence staining and radioimmunoprecipitation of biosynthetically labeled proteins, mesangial cells express laminin beta1, beta2, and gamma1 chains. Mesangial cells do not express laminin alpha1 or alpha2. MC produce a unique alpha chain, designated alpha'm. These laminin chains assemble into two major isoforms. One contains alpha'mbeta1gamma1, co-precipitates with entactin and is assembled into the fibrillar extracellular matrix. The second isoform contains alpha'mbeta2 and a presumed gamma chain that migrates in gel slightly ahead of gamma1. The beta2-containing isoform is concentrated in punctate sites on the cell surface. In addition, mesangial cells display different phenotypes when plated on laminin-1 (alpha1beta1gamma1), as compared to purified beta2. An LRE-containing peptide of laminin beta2 serves as an attachment site for mesangial cells and is sufficient to induce the phenotype observed with intact beta2. These data suggest that laminin isoform expression plays an important role in mesangial cell phenotype and function.     

Alpha 6 beta 4 integrin and newly deposited laminin-1 and laminin-5 form the adhesion mechanism of gastric carcinoma. Continuous expression of laminins but not that of collagen VII is preserved in invasive parts of the carcinomas: implications for acquisition of the invading phenotype

We studied the expression and distribution of different laminin chains, the alpha 6 beta 4 integrin and type VII collagen, i.e., components of the epithelial adhesion complex, in gastric carcinomas and in suggested preneoplastic stages of this malignancy. Intestinal-type gastric carcinomas showed strong reactivity for laminin alpha 1, alpha 3, beta 1, and beta 3 chains, the components of laminin-1 and -5, at the interface between malignant cells and tumor stroma. The reactivities were continuous throughout the carcinomas, even in structures invading through the smooth muscle layers of the gastric wall. The expression of different laminin chains was accompanied by strong polarized reactivity for the alpha 6 beta 4 integrin, which is a receptor for both laminin-1 and laminin-5. Collagen type VII was only occasionally present at sites showing reactivity for laminin-5 and was totally absent from the cell islands invading through the gastric wall. Intestinalized gastric epithelium showed a similar expression pattern of laminins and the alpha 6 beta 4 integrin as the gastric carcinomas. Our results suggest that gastric carcinomas use the alpha 6 beta 4 integrin and newly deposited laminin-1 and -5, accompanied by the disappearance of type VII collagen, as their mechanism of adhesion during the invasion through surrounding tissues. Unlike in previous studies, the reactivity for the laminin-5 protein was not restricted to the invading cells but surrounded the malignant glandular structures throughout the tumor. Our results also show that both intestinal-type gastric carcinoma, and intestinal metaplasia mimic the gastric surface epithelium in the expression pattern of laminins and the beta 4 integrin subunit. This supports previous studies proposing a pathogenetic sequence from intestinal metaplasia to gastric carcinoma.     

Identification of integrin-dependent and -independent cell adhesion domains in COOH-terminal globular region of laminin-5 alpha 3 chain

Laminin-5 is an isoform of laminin that consists of alpha 3, beta 3, and gamma 2 chains and has potent cell adhesion- and cell migration-promoting activities. In this study, five subdomains in the COOH-terminal globular (G) domain of human laminin alpha 3 chain were individually expressed in Escherichia coli, and their biological activities were investigated. Recombinant G2, G4, and G5 domains promoted adhesion to plastic plates of HT1080 fibrosarcoma cells, A431 epidermoid carcinoma cells, and ECV304 vascular endothelial cells. For the cell adhesion activity, the G2 domain required a divalent cation and heat-sensitive conformation more strongly than G4 and G5. The cell adhesion to G2 but not G4 and G5 was effectively inhibited by an anti-integrin alpha 3 antibody. A cell adhesion sequence of 22 amino acids, alpha 3G2A, that was homologous to the integrin alpha 3 beta 1-binding sequence GD-6 of laminin alpha 1 chain was identified within the G2 structure. The cell adhesion to alpha 3G2A peptide was also inhibited by the anti-integrin alpha 3 antibody. The cell adhesion to G2, alpha 3G2A, G4, and G5 was strongly inhibited by heparin, but that to native laminin-5 was inhibited less effectively. Moreover, G5 potently stimulated chemotactic migration of rat liver epithelial cells in Boyden chambers, but G2 and G4 did not. These results indicate that the G domain of laminin alpha 3 contains multiple cell binding sites with different mechanisms and different functions. The G2 domain seems to recognize integrin alpha 3 beta 1, whereas G4 and G5 may interact with heparin-like molecules on cell surface.     

Laminin localization in enterocytic basement membrane of rat small bowel grafts. A light and electron microscopic study

In vitro laminins stimulate numerous biological effects, such as cell migration, proliferation, attachment and differentiation. In vitro laminins influence immunocompetent cells and in vivo possibly play an important role in graft rejection. To establish how laminins could be involved in the regulation of acute rejection of small bowel allografts (with and without immunosuppression), we investigated laminin distribution in rat small bowel allografts four days after transplantation, i.e., before the onset of histological signs of rejection, using antibodies against alpha1, beta1, gamma1 chain of laminin-1. In immunosuppressed allografts, the ultrastructure of the enterocytic basement membrane appeared normal, but no laminin staining was seen in this membrane, although basement membranes of intramural blood vessels and muscle cells were normally stained. In non-operated immunosuppressed rats, laminin staining was clearly reduced in the enterocytic basement membrane, demonstrating that cyclosporin A is able to affect this membrane. Since only rats in which laminin is altered survive, this laminin alteration in the enterocytic basement membrane presumably plays an important role in overcoming the acute rejection.     

Rapid expression and specific localization of tenascin in gastric ulcer healing in rats

This study was done to investigate the gene expression and localization of tenascin in ulcerated gastric tissues during the healing process with Northern blot analysis and immunohistochemical technique. Gastric ulcers in rats were produced by acetic acid. Tenascin mRNA levels in the ulcerated tissue were significantly increased in a biphasic manner (12 h and day 5), preceding the increase in collagen type IV and laminin mRNA levels, and returned to control levels on day 11. In intact tissues, tenascin was mainly localized in the basement membrane above the proliferative zone, in contrast to the predominant localization of collagen type IV and laminin below the proliferative zone. On the ulcer margin from 12 h to day 5, tenascin was abundantly observed in the lamina propria around nonproliferating new epithelial cells, but collagen type IV and laminin were not seen in this lamina propria. On day 7, tenascin, expressed in the lamina propria, was replaced by collagen type IV and laminin. Thus, the rapid expression and unique localization of tenascin suggest the important role of tenascin in gastric ulcer healing.     

Vaginal intraepithelial neoplasia (VAIN) following hysterectomy in patients treated for carcinoma in situ of the cervix

BACKGROUND: Cell adhesion in the limbal region is of outstanding importance for the regeneration of the corneal epithelium and for repair mechanisms after antiglaucomatous fistulating surgery. In the basement membranes cell adhesion is largely modified by the extracellular matrix protein laminin. The aim of our study was to establish the immunohistochemical pattern of the different laminin-isoforms and subunits in the basal membrane of the limbal conjunctiva and episcleral vessels. MATERIAL AND METHOD: For immunohistochemistry five normal human donor eyes were included; we used antibodies against the laminin heterotrimers 1 and 2, against the laminin subunits alpha 2, beta 1, beta 2, gamma 1, gamma 2 and against the laminin-associated protein nidogen. RESULTS: The basement membrane of the limbal conjunctiva reveals immunoreactivity against all used antibodies. The subconjunctival and episcleral vessels showed no staining for the laminin subunit gamma 2, but for all other used antibodies. CONCLUSION: The basement membrane of the limbal and conjunctival epithelium as well as the basement membrane of subconjunctival and episcleral vessels express a broad spectrum of laminin variants. This diversity emphasizes functional specialization of the limbal region, although the exact importance of the laminin variants is still unknown.     

Differentiation-specific enhancer activity in transduced keratinocytes: a model for epidermal gene therapy

Active sequences from the laminin alpha1 and alpha2 chain carboxyl-terminal globular domains (G domain) have been identified by screening overlapping synthetic peptides in a number of biological assays (Nomizu et al. [1995] J. Biol. Chem. 270:20583-20590; Nomizu et al. [1996] FEBS Lett. 396:37-42). We have tested the activity of these peptides in submandibular gland explants of embryonic day 13 mice to determine the functional sites involved in organ development. The laminin alpha1 chain peptide, RKRLQVQLSIRT (residues 2719-2730 and designated AG-73), significantly inhibited epithelial branching morphogenesis. In contrast, other cell adhesive laminin alpha1 chain peptides including the AASIKVAVSADR and NRWHSIYITRFG failed to inhibit the branching. MG-73, a homologue of AG-73 from the laminin alpha2 chain, did not inhibit the branching. The alpha2 chain peptide had no effect, which may be due to the low levels of this laminin chain in day 13 mice. Laminin alpha2 chain-specific monoclonal antibodies strongly reacted with the basement membranes of developed acini but only weakly stained embryonic day 13 submandibular epithelium. The expression of E-cadherin and alpha6 integrin, as detected by immunofluorescence, were unchanged in both AG-73 and control scramble peptide-treated epithelial cells of the explants. In contrast, immunostaining of nidogen/entactin showed that explants treated with AG-73 for 3 days had a discontinuous basement membrane. Explants treated for 3 days with control peptide showed a normal basement membrane. These results suggest that the region containing the AG-73 sequence of the laminin alpha1 chain is crucial for development of submandibular gland at early embryonic stages. The discontinuous basement membrane in AG-73-treated explants may indicate an important role for this region in basement membrane assembly.     

Differential effects of low and high concentrations of 4-aminopyridine on axonal conduction in normal and injured spinal cord

Laminins, the main components of basement membranes, are heterotrimers consisting of alpha, beta, and gamma polypeptide chains linked together by disulfide bonds. Laminins-1 and -2 are both composed of beta1 and gamma1 chains and differ from each other on their alpha chain, which is alpha1 and alpha2 for laminin-1 and -2, respectively. The present study shows that whereas laminins-1 and -2 are synthesized in the mouse developing lung and in epithelial-mesenchymal cocultures derived from it, epithelial and mesenchymal monocultures lose their ability to synthesize the laminin alpha1 chain. Synthesis of laminin alpha1 chain however returns upon re-establishment of epithelial-mesenchymal contact. Cell-cell contact is critical, since laminin alpha1 chain is not detected in monocultures exposed to coculture-conditioned medium or in epithelial-mesenchymal cocultures in which heterotypic cell-cell contact is prevented by an interposing filter. Immunohistochemical studies on cocultures treated with brefeldin A, an inhibitor of protein secretion, indicated both epithelial and mesenchymal cells synthesize laminin alpha1 chain upon heterotypic cell- cell contact. In a set of functional studies, embryonic lung explants were cultured in the presence of monoclonal antibodies to laminin alpha1, alpha2, and beta/gamma chains. Lung explants exposed to monoclonal antibodies to laminin alpha1 chain exhibited alterations in peribronchial cell shape and decreased smooth muscle development, as indicated by low levels of smooth muscle alpha actin and desmin. Taken together, our studies suggest that laminin alpha1 chain synthesis is regulated by epithelial-mesenchymal interaction and may play a role in airway smooth muscle development.     

Regulation of the beta 2 subunit chain of laminin in developing rabbit fetal lung tissue

Laminins are a family of basement membrane-associated heterotrimeric proteins that are important in mediating the growth, migration, and differentiation of a variety of cell types. The beta 2 subunit chain is a component of several laminin isoforms, e.g., laminin-3, laminin-4, laminin-7, and possibly other, as yet uncharacterized laminin isoforms. Utilizing monoclonal antibodies directed against the beta 2 subunit chain of laminin, we detected this protein in fetal, neonatal, and adult lung tissues. The relative amount of laminin beta 2 subunit chain in fetal lung tissue increased as gestation proceeded, reaching its peak around the time of alveolar type II cell differentiation in the rabbit. The laminin beta 2 subunit chain was localized in early gestational age rabbit fetal lung tissue primarily in basement membranes of prealveolar ducts, airways, and smooth muscle cells of airways and arterial blood vessels. A rabbit laminin beta 2 cDNA was generated using RT-PCR and utilized as a probe in northern blot analysis to characterize the levels of laminin beta 2 mRNA in developing rabbit lung tissue. Similar to the pattern of laminin beta 2 protein induction observed in fetal lung tissue, laminin beta 2 mRNA levels were maximal late in gestation. Utilizing a laminin beta 2 chain cRNA probe and in situ hybridization, we detected laminin beta 2 mRNA in the epithelial cells of prealveolar ducts, the alveolar wall, and airways, as well as in connective tissue cells, and the smooth muscle cells of airways and blood vessels in fetal and adult lung tissues. In addition, using an in vitro explant model, we determined that alveolar type II cells are capable of synthesizing laminin beta 2 subunit mRNA and depositing this laminin subunit chain in the basement membrane beneath type II cells. The results of this study are suggestive that the laminin beta 2 chain may be involved in alveolar epithelial cell differentiation.