Steady-state levels of mRNAs coding for the type IV collagen and laminin polypeptide chains of basement membranes exhibit marked tissue-specific stoichiometric variations in the rat

Rat retina, lens, and kidney from 8-week-old animals were assayed for the steady-state levels of mRNAs for four basement membrane components: The alpha 1 chain of type IV collagen, the alpha 2 chain of type IV collagen, the B1 chain of laminin, and the B2 chain of laminin. Each tissue exhibited markedly different ratios of the four mRNAs. The mRNA ratio for the alpha 1 chain of type IV collagen to the B1 chain of laminin varied from a value of 0.7 in retina to a value of 17 in lens. Also, the mRNA ratio for the alpha 1 chain to the alpha 2 chain of type IV collagen varied from 1.6 in retina to 17 in lens, and the mRNA ratio for the B1 chain to the B2 chain of laminin varied from 0.6 in lens to 2.9 in kidney. The mRNA coding for the alpha 1 chain of type IV collagen decreased in all three tissues as the animals increased in age from 8 to 16 weeks, with the rate of decline being greater in retina than in lens of kidney. The levels of mRNA coding for the B1 and the B2 chains of laminin decreased in the kidney between 8 and 16 weeks but at different rates. Comparison of mRNAs from kidney of rats over this time period showed that the ratio of alpha 1 to B1 remained relatively constant with age, whereas the ratio of B1 to B2 increased. One possible explanation for the results is that each tissue has elaborate, tissue-specific controls for translation that provide synthesis of basement membrane components in the same proportion, in spite of the varying steady-state levels of the mRNAs. A more likely explanation is that different tissues synthesize type IV collagen and laminin at different rates, and that even the subunit compositions of the type IV collagen and laminin molecules vary from tissue to tissue and in an age-dependent manner.     

Increased steady-state levels of laminin B1 mRNA in kidneys of long-term streptozotocin-diabetic rats. No effect of an aldose reductase inhibitor

The steady-state levels of mRNAs coding for two components of basement membranes, the alpha 1 chain of type IV collagen and the B1 chain of laminin, were measured in the kidneys of male CDF rats following the induction of diabetes with streptozotocin for periods of between 2 days and 28 weeks. The concentration of mRNA for the alpha 1 chain of type IV collagen/microgram of RNA decreased markedly with age in control and diabetic rats. The diabetic level was significantly lower than control after 2 and 11 weeks of diabetes. After 28 weeks, however, there was no significant difference from the levels in control animals. Treatment of control and diabetic rats with the aldose reductase inhibitor Statil (350 mg/kg diet) did not affect the levels of the mRNA for the alpha 1 chain of type IV collagen. In contrast to the continuous decline in the concentration of mRNA for the alpha 1 chain of type IV collagen, the level of mRNA for the B1 chain of laminin increased two-fold between 11 and 28 weeks after induction of diabetes. This increase occurred as aging of control rats reduced the level of laminin B1 mRNA by approximately 50%. Treatment with Statil had no effect on laminin B1 mRNA levels. In control rats there was no change in the ratio of the levels of mRNAs for laminin B1: alpha 1 (IV) collagen with age. The mean ratio was 0.97 +/- 0.10 at 19 weeks and 1.0 +/- 0.10 at 36 weeks of age. In diabetic rats there was a marked increase in the ratio from 0.85 +/- 0.11 at 19 weeks to 3.2 +/- 1.2 at 36 weeks of age. The increased abundance of mRNA for laminin B1 raises the possibility that increased synthesis of laminin contributes to the thickening and abnormal function of renal basement membranes in streptozotocin-diabetic rats.     

Sex hormone-binding globulin, androgen-binding protein, and vitamin K-dependent protein S are homologous to laminin A, merosin, and Drosophila crumbs protein.

Androgen-binding protein (ABP) and sex hormone-binding globulin (SHBG) are extracellular steroid-binding proteins that are homologous to the COOH-terminal domain of vitamin K-dependent protein S, a protein important in blood clotting. We find that the sequences of ABP, SHBG, and protein S are also similar to two basement membrane proteins, laminin and merosin, and to an integral membrane protein, Drosophila crumbs protein. These latter three proteins have important roles in regulating differentiation and development. The sequence similarity corresponds to the G domain of laminin A chain, which binds heparin and type IV collagen. Analysis of a multiple alignment of these proteins reveals one well-conserved segment corresponding to the part of SHBG that binds to its membrane receptor and another corresponding to the part of protein S that binds to C4b-binding protein. The similarities suggest that ABP, SHBG, and protein S may also have functions related to that of laminin and merosin.     

Genes for basement membrane proteins are coordinately expressed in differentiating F9 cells but not in normal adult murine tissues

We have obtained cDNA clones coding for the A, B1, and B2 chains of laminin by screening a cDNA library prepared from mouse EHS tumor poly(A)RNA in the lambda gt11 expression vector with polyclonal antibody against denatured laminin. These cDNA clones were used in combination with a cDNA clone coding for the alpha 1 type IV collagen chain to study the regulation of genes for these basement membrane proteins in retinoic acid-induced differentiating mouse F9 teratocarcinoma cells and in various adult murine tissues. The levels of mRNA for the laminin A, B1, and B2 chains and for the alpha 1 type IV collagen chain were increased simultaneously and reached a maximum at almost the same time during the differentiation of F9 cells, suggesting coordinate expression in these cells. The tissue levels of mRNA encoding for the basement membrane components, however, varied considerably. The highest level of the B1 chain mRNA was observed in kidney, whereas, the levels of mRNA for A and B2 chains were highest in heart. Almost the same levels of expression of the alpha 1(IV) collagen mRNA were found in kidney, lung, and heart. The results indicate that the expression of genes for the basement membrane proteins is not coordinately regulated in these tissues. It is thus possible that different subunit structures of the laminin molecule may exist in tissues.     

Interactions of basement membrane components

The binding of laminin, type IV collagen, and heparan sulfate proteoglycan to each other was assessed. Laminin binds preferentially to native type IV (basement membrane) collagen over other collagens. A fragment of laminin (Mr 600 000) containing the three short chains (Mr 200 000) but lacking the long chain (Mr 400 000) showed the same affinity for type IV collagen as the intact protein. The heparan sulfate proteoglycan binds well to laminin and to type IV collagen. These studies show that laminin, type IV collagen and heparan sulfate proteoglycan interact with each other. Such interactions in situ may determine the structure of basement membranes.     

The incubation of laminin, collagen IV, and heparan sulfate proteoglycan at 35 degrees C yields basement membrane-like structures

Three basement membrane components, laminin, collagen IV, and heparan sulfate proteoglycan, were mixed and incubated at 35 degrees C for 1 h, during which a precipitate formed. Centrifugation yielded a pellet which was fixed in either potassium permanganate for ultrastructural studies, or in formaldehyde for Lowicryl embedding and immunolabeling with protein A-gold or anti-rabbit immunoglobulin-gold. Three types of structures were observed and called types A, B, and C. Type B consisted of 30-50-nm-wide strips that were dispersed or associated into a honeycomb-like pattern, but showed no similarity with basement membranes. Immunolabeling revealed that type B strips only contained heparan sulfate proteoglycan. The structure was attributed to self-assembly of this proteoglycan. Type A consisted of irregular strands of material that usually accumulated into semisolid groups. Like basement membrane, the strands contained laminin, collagen IV, and heparan sulfate proteoglycan, and, at high magnification, they appeared as a three-dimensional network of cord-like elements whose thickness averaged approximately 3 nm. But, unlike the neatly layered basement membranes, the type A strands were arranged in a random, disorderly manner. Type C structures were convoluted sheets composed of a uniform, dense, central layer which exhibited a few extensions on both surfaces and was similar in appearance and thickness to the lamina densa of basement membranes. Immunolabeling showed that laminin, collagen IV, and proteoglycan were colocalized in the type C sheets. At high magnification, the sheets appeared as a three-dimensional network of cords averaging approximately 3 nm. Hence, the organization, composition, and ultrastructure of type C sheets made them similar to the lamina densa of authentic basement membranes.     

Basement membrane procollagen IV and its specialized carboxyl domain are conserved in Drosophila, mouse, and human

Interaction with the extracellular matrix is important for the proliferation and differentiation of cells during development. A specialized extracellular matrix, basement membrane, is built around a scaffold of procollagen IV molecules. We report the sequence of a 2.5-kilobase cDNA which contains the carboxyl end of a Drosophila melanogaster procollagen IV. The amino acid sequence of the carboxyl-terminal domain, which forms an essential intermolecular linkage between procollagen IV molecules, is 59% identical in Drosophila and vertebrate procollagens IV, and an additional 17% of residues are conservatively substituted. This implies that the nature of the linkage is also conserved. We suggest that intermolecular junctions through procollagen IV carboxyl domains are fundamental elements of the molecular architecture of Metazoan basement membranes and have been conserved during evolution. The isolation and identification of this basement membrane collagen gene of Drosophila will help in deducing the function of procollagen IV in basement membranes.     

Interactions of basement membrane components

The binding of laminin, type IV collagen, and heparan sulfate proteoglycan to each other was assessed. Laminin binds preferentially to native type IV (basement membrane) collagen over other collagens. A fragment of laminin (M_r 600 000) containing the three short chains (M_r 200 000) but lacking the long chain M_r 400 000) showed the same affinity for type IV collagen as the intact protein. The heparan sulfate proteoglycan binds well to laminin and to type IV collagen. These studies show that laminin, type IV collagen and heparan sulfate proteoglycan interact with each other. Such interactions in situ may determine the structure of basement membranes.     

Laminin promotes formation of cord-like structures by Sertoli cells in vitro

Basement membranes are thin extracellular matrices which contact epithelial cells and promote their adhesion, migration, differentiation, and morphogenesis. These matrices are composed of collagen IV, heparan sulfate proteoglycan, laminin, and entactin as well as other minor components. Sertoli cells, like most epithelial cells, are in contact at their basal surface with a basement membrane. When cultured within three-dimensional basement membrane gels (Matrigel), Sertoli cells reorganize into cords that resemble testicular seminiferous cords found in the in vivo differentiating testis. Anti-laminin and anti-entactin antisera inhibit this cord morphogenesis by Sertoli cells whereas antisera against type IV and type I collagen, heparan sulfate proteoglycan, fibronectin, and preimmune sera had no effect. The RGD (RGDS-NH2) sequence, found in the cell binding domain of the integrin family of cell adhesion molecules as well as in the A chain of laminin and in entactin, effectively inhibited Sertoli cell cord formation at a concentration of 1.0 mg/ml but was unable to prevent Sertoli cell attachment at concentrations as high as 2.0 mg/ml. A synthetic pentapeptide from a cell-binding domain of the B1 chain of laminin. YIGSR-NH2, inhibited cord formation at a concentration of 0.25 mg/ml, but Sertoli cells were still adherent to the basement membrane matrix. At concentrations greater than 0.50 mg/ml, Sertoli cells detached. Antiserum against the YIGSR-NH2-containing sequence was also effective in inhibiting cord formation by Sertoli cells. Ligand (YIGSR-NH2 peptide) blot analysis of Sertoli cell lysates revealed an interaction with a major band at 60 kDa and with minor bands at 39 and 127 kDa. Furthermore, in Western blot analysis the anti-67-kDa laminin-binding protein antibody recognized a 59- to 60-kDa protein in Sertoli cells. The data indicate that laminin is involved in both Sertoli cell attachment and migration during formation of histotypic cord structures by these cells in culture. Two separate laminin cell-binding domains appear to be involved in Sertoli cell cord morphogenesis in vitro and are likely to participate in the formation of seminiferous cords in vivo.     

Localization of binding sites for laminin, heparan sulfate proteoglycan and fibronectin on basement membrane (type IV) collagen

Rotary shadowing electron microscopy was used to examine complexes formed by incubating combinations of the basement membrane components: type IV collagen, laminin, large heparan sulfate proteoglycan and fibronectin. Complexes were analyzed by length measurement from the globular (COOH) domain of type IV collagen, and by examination of the four arms of laminin and the two arms of fibronectin. Type IV collagen was found to contain binding sites for laminin, heparan sulfate proteoglycan and fibronectin. With laminin the most frequent site was centered approximately 81 nm from the carboxy end of type IV collagen. Less frequent sites appeared to be present at approximately 216 nm and approximately 291 nm, although this was not apparent when the sites were expressed as a fraction of the length of type IV collagen to which they were bound. For heparan sulfate proteoglycan the most frequent site occurred at approximately 206 nm with a less frequent site at approximately 82 nm. For fibronectin, a single site was present at approximately 205 nm. Laminin bound to type IV collagen through its short arms, particularly through the end of the lateral short arms and to heparan sulfate proteoglycan mainly through the end of its long arm. Fibronectin bound to type IV collagen through the free end region of its arms. Using a computer graphics program, the primary laminin binding sites of two adjacent type IV collagen molecules were found to align in the "polygonal" model of type IV collagen, whereas with the "open network" model, a wide meshed matrix is predicted. It is proposed that basement membrane may consist of a lattice of type IV collagen coated with laminin, heparan sulfate proteoglycan and fibronectin.     

Vitamin A deficiency disturbs collagen IV and laminin composition and decreases matrix metalloproteinase concentrations in rat lung. Partial reversibility by retinoic acid

Vitamin A is essential for lung development and pulmonary cell differentiation. Its deficiency leads to altered lung structure and function and to basement membrane architecture and composition disturbances. Previously, we showed that lack of retinoids thickens the alveolar basement membrane and increases collagen IV, which are reversed by retinoic acid, the main biologically active vitamin A form. This study analyzed how vitamin A deficiency affects the subunit composition of collagen IV and laminin of lung basement membranes and pulmonary matrix metalloproteinase content, plus the recovering effect of all-trans-retinoic acid. Male weanling pups were fed a retinol-adequate/-deficient diet until 60 days old. A subgroup of vitamin-A-deficient pups received daily intraperitoneal all-trans-retinoic acid injections for 10 days. Collagen IV and laminin chain composition were modified in vitamin-A-deficient rats. The protein and mRNA contents of chains α1(IV), α3(IV) and α4(IV) increased; those of chains α2(IV) and α5(IV) remained unchanged; and the protein and mRNA contents of laminin chains α5, β1 and γ1 decreased. The mRNA of laminin chains α2 and α4 also decreased. Matrix metalloproteinases 2 and 9 decreased, but the tissue inhibitors of metalloproteinases 1 and 2 did not change. Treating vitamin-A-deficient rats with retinoic acid reversed all alterations, but laminin chains α2, α4 and α5 and matrix metalloproteinase 2 remained low. In conclusion, vitamin A deficiency alters the subunit composition of collagen IV and laminin and the lung's proteolytic potential, which are partly reverted by retinoic acid. These alterations could contribute to impaired lung function and predispose to pulmonary disease.     

Expression of extracellular matrix components is regulated by substratum

Reconstituted basement membranes and extracellular matrices have been demonstrated to affect, positively and dramatically, the production of milk proteins in cultured mammary epithelial cells. Here we show that both the expression and the deposition of extracellular matrix components themselves are regulated by substratum. The steady-state levels of the laminin, type IV collagen, and fibronectin mRNAs in mammary epithelial cells cultured on plastic dishes and on type I collagen gels have been examined, as has the ability of these cells to synthesize, secrete, and deposit laminin and other, extracellular matrix proteins. We demonstrate de novo synthesis of a basement membrane by cells cultured on type I collagen gels which have been floated into the medium. Expression of the mRNA and proteins of basement membranes, however, are quite low in these cultures. In contrast, the levels of laminin, type IV collagen, and fibronectin mRNAs are highest in cells cultured on plastic surfaces, where no basement membrane is deposited. It is suggested that the interaction between epithelial cells and both basement membrane and stromally derived matrices exerts a negative influence on the expression of mRNA for extracellular matrix components. In addition, we show that the capacity for lactational differentiation correlates with conditions that favor the deposition of a continuous basement membrane, and argue that the interaction between specialized epithelial cells and stroma enables them to create their own microenvironment for accurate signal transduction and phenotypic function.     

Drosophila basement membrane procollagen IV. I. Protein characterization and distribution

A collagen was isolated from Drosophila E85, Schneider line 2L and Kc cell cultures. The purified protein was characterized and antibodies were raised against it. Immunofluorescence microscopy locates this material to the regions of basement membranes of Drosophila embryos, larvae, and adults. The molecules are mostly, or entirely, homotrimers of one polypeptide chain linked by interchain disulfide bonds. The partial amino acid sequences of a cyanogen bromide cleavage product of this chain are identical with a part of the virtual translation product of the Drosophila pro alpha 1(IV) nucleotide sequence that is reported in the accompanying paper. This gene is at Drosophila chromosome location 25C and was identified by the high homology of one part of it with the noncollagenous carboxyl terminus (NC1) of vertebrate type IV basement membrane collagens (Blumberg, B., MacKrell, A. J., Olson, P. F., Kurkinen, M., Monson, J. M., Natzle, J. E., and Fessler, J. H. (1987) J. Biol. Chem. 262, 5947-5950). In the electron microscope each molecule appears as a thread with a knob at one end, which contains the carboxyl peptide domains. The variation of flexibility of the thread was mapped along its length. Pulse-chase labeling of cell cultures showed that these molecules associate into disulfide-linked dimers and higher oligomers that can be partly separated by velocity sedimentation and are resolved by sodium dodecyl sulfate-agarose gel electrophoresis. Dimers and higher oligomers formed by overlap of the amino ends of molecules were found. Mild pepsin digestion of Drosophila embryos and larvae solubilized the corresponding disulfide-linked collagen molecules, and Staphylococcus aureus V8 protease peptide maps showed the identity of the collagen derived from animals and from cell cultures. Individual, native molecules have a sedimentation coefficient s20,w = 4.1 S, the dichroic spectrum and amino acid composition of a collagen, and a Tm = 31 degrees C. Positive in situ hybridization with a specific probe for this collagen began 6-8 h after egg laying and showed message in the locations of embryos and larvae which reacted with the antibodies. This included some prominent individual cells in the hemolymph.     

Modified basement membrane composition during bronchopulmonary tumor progression.

During tumor progression, the extracellular matrix (ECM) and particularly the basement membrane (BM) appear to be dynamic structures that are not only degraded but also deposited around tumor clusters. In this study we examined by immunohistochemistry the localization of three chains of Type IV collagen (alpha1, alpha3 and alpha5), Type VII collagen, and laminin 5 at different stages of bronchopulmonary cancers. In normal tissues, alpha1(IV) chain was detected in all BMs (bronchial, vascular, alveolar, and glandular), alpha5(IV) chain was present only in vascular BM, and laminin 5 and Type VII collagen were co-localized in bronchial and glandular BMs, whereas alpha3(IV) immunolabeling was totally absent from normal bronchi. In well-differentiated carcinomas, alpha3(IV) chain staining was found in some neosynthetized BMs interfacing the tumor cell and the stromal compartment, contrasting with the total absence of labeling in normal tissues. alpha1(IV) chain showed strong reactivity in all BM. Laminin 5 and Type VII collagen were also detected in neosynthetized BM. In poorly differentiated invasive cancers, alpha3(IV) chain and Type VII collagen were not found, whereas laminin 5 and alpha1(IV) chain persisted. The most important modifications in BM composition during tumor progression therefore appear to be the appearance of the alpha3 (IV) chain in well-differentiated carcinomas and its subsequent disappearance in poorly differentiated carcinomas, together with the loss of type VII collagen. alpha5(IV) chain distribution was restricted in vascular BM of well- and poorly differentiated carcinomas. These results show that the composition of BM is modified during the progression of bronchopulmonary tumor, emphasizing that the BM represents a dynamic element in tumor progression and has an important role in tumor cell invasiveness.     

Laminin B1 and Collagen Type IV Gene Expression in Transected Peripheral Nerve: Reinnervation Compared to Denervation

The expression of B1 laminin and type IV collagen was followed in the microsurgically isolated endoneurium of transected rat sciatic nerves from 3 days until 8 weeks. Northern hybridizations revealed that after nerve transection the proximal stumps of denervated, as well as freely regenerating, nerves showed a markedly increased expression of laminin and type IV collagen which lasted from 3 days up to 8 weeks. In the distal stumps, close to the site of transection (2-7 mm), the expression of laminin, and to a certain extent that of type IV collagen, seemed to be enhanced if free axonal reinnervation was allowed. Further distally (10-15 mm), the patterns of B1 laminin and type IV collagen expression were similar in both experimental groups, so that an increased expression was noticed during the first 2 weeks. The present results suggest that laminin and type IV collagen gene expression is markedly different in different parts of transected rat sciatic nerve. During peripheral nerve regeneration, there is a long-lasting basement membrane gene expression in the proximal stump. In the distal part of the transected nerve, the axonal reinnervation possibly up-regulates, but is not essential for, the expression of B1 laminin and type IV collagen.     

Collagen IV alpha 3, alpha 4, and alpha 5 chains in rodent basal laminae: sequence, distribution, association with laminins, and developmental switches

Collagen IV is a major component of vertebrate basal laminae (BLs). Studies in humans have revealed a family of genes encoding alpha 1- alpha 6 collagen IV chains and implicated alpha 3-alpha 6 in disease processes (Goodpasture and Alport syndromes and diffuse leiomyomatosis). To extend studies of these components to an experimentally accessible animal, we cloned cDNAs encoding partial collagen alpha 3, alpha 4, and alpha 5(IV) chains from the mouse. Ribonuclease protection assays showed that all three genes were expressed at highest levels in kidney and lung; alpha 5(IV) was also expressed at high levels in heart. We then made antibodies specific for each collagen IV chain. Immunohistochemical studies of several tissues revealed many combinations of collagen IV chains; however, alpha 3 and alpha 4 (IV) were always coexpressed, and only appeared in BLs that were alpha 5(IV) positive. The alpha 3-alpha 5(IV) chains were frequently but not exclusively associated with the S (beta 2) chain of laminin, as were the alpha 1, 2 (IV) collagen chains with laminin B1 (beta 1). An analysis of developing rat kidney BLs showed that newly formed (S-shaped) nephrons harbored collagen alpha 1 and alpha 2(IV) and laminin B1; maturing (capillary loop stage) BLs contained collagen alpha 1-alpha 5(IV) and laminin B1 and S-laminin; and mature glomerular BLs contained mainly collagen alpha 3-alpha 5(IV) and S-laminin. Thus, collagen alpha 1 and alpha 2(IV) and laminin B1 appear to be fetal components of the glomerular BL, and there is a developmental switch to collagen alpha 3-alpha 5(IV) and S-laminin expression.