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.     

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.     

In situ hybridization reveals temporal and spatial changes in cellular expression of mRNA for a laminin receptor, laminin, and basement membrane (type IV) collagen in the developing kidney

The appearance of extracellular matrix molecules and their receptors represent key events in the differentiation of cells of the kidney. Steady-state mRNA levels for a laminin receptor, the laminin B1, B2, and A chains, and the alpha 1-chain of collagen IV (alpha 1[IV]), were examined in mouse kidneys at 16 d gestation and birth, when cell differentiation is active, and 1-3 wk after birth when this activity has subsided. Northern analysis revealed that mRNA expression of laminin receptor precedes the alpha 1(IV) and laminin B chains whereas laminin A chain mRNA expression was very low. In situ hybridization reflected this pattern and revealed the cells responsible for expression. At 16 d gestation, laminin receptor mRNA was elevated in cells of newly forming glomeruli and proximal and distal tubules of the nephrogenic zone located in the kidney cortex. These cells also expressed mRNA for alpha 1(IV) and laminin chains. At birth, mRNA expression of receptor and all chains remained high in glomeruli but was reduced in proximal and distal tubules. At 1 wk after birth, expression was located in the medulla over collecting ducts and loops of Henle. Little expression was detectable by 3 wk. These results suggest that cellular expression of steady-state mRNA for laminin receptor, laminin, and collagen IV is temporally linked, with laminin receptor expression proceeding first and thereafter subsiding.     

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.     

Laminin A chain: expression during Drosophila development and genomic sequence.

A Drosophila laminin A chain gene was characterized as a 14 kb genomic nucleotide sequence which encodes an open reading frame of 3712 amino acids in 15 exons. Overall, this A chain is similar to its vertebrate counterparts, especially in its N- and C-terminal globular domains, but the sequence that forms the laminin A short arm is quite different and larger. Laminin messages appear in newly formed mesoderm and are later prominently expressed in hemocytes, which also synthesize basement membrane collagen IV. The composition of Drosophila basement membranes changes with development. A novel method of tandemly fused RNA probes showed that developmental increases of laminin mRNAs were primarily associated with periods of morphogenesis, and preceded those of collagen IV, a protein strongly expressed during growth. The ratio of A:B1:B2 mRNAs varied little during embryogenesis, with less mRNA for A than B chains. Staining of embryos with antibodies confirmed and extended the information provided by in situ hybridization. Homologs of the G-subdomains of this A chain, which occur in interacting regions of agrin, perlecan, laminin and sex steroid binding protein, may be involved in protein associations.     

Collagen synthesis by long-lived mRNA in embryonic chicken lens

Lens capsule collagen synthesis by epithelial and fiber cells was examined by immunoprecipitation and collagenase digestion in embryonic and posthatch chicken eye lens. Epithelial cells and lens fibers in the process of terminal differentiation produce alpha 1 and alpha 2 type IV collagen chains. At 6 days of embryonic development in addition to the alpha 1 (IV) and alpha 2 (IV) collagen chains, lens cells produce high molecular weight collagenase-sensitive proteins not immunologically related to type IV collagen. Lens capsule collagen components have been identified in central and outer fibers isolated from 18-day embryos and from 10-day posthatch chicken eyes. At these stages, fibers which have an increasing number of picnotic nuclei still show collagen synthesis due to long-lived mRNA. Analysis of collagen synthesis by lens cells incubated with actinomycin D suggests that stabilization of collagen mRNA occurs in lens fiber cells and to a lesser extent in epithelial cells as early as 6 days of embryonic development.     

The integrin beta 1 subunit associates with the vitronectin receptor alpha v subunit to form a novel vitronectin receptor in a human embryonic kidney cell line.

We describe a novel integrin heterodimer on the surface of the human embryonic kidney cell line 293. This receptor is comprised of alpha v and beta 1 subunits, each of which has been previously found in association with other integrin subunits. This alpha v.beta 1 complex was identified as the predominant vitronectin receptor (VnR) on the surface of 293 cells by immunoprecipitation with antibodies raised against the alpha v subunit. Polymerase chain reaction analysis detected mRNAs for alpha v and beta 1 subunits while no evidence was obtained for beta 2, beta 3, or alpha IIb integrin subunit mRNA. Immunoprecipitation of surface-iodinated proteins with antibodies to alpha v gave bands of 150 and 120 kDa. The 120-kDa band reacted with antibodies to beta 1 in immunoblotting experiments. 293 cells adhere to vitronectin, fibronectin, laminin, and collagen IV, while von Willebrand factor and fibrinogen, known ligands of the VnR (alpha v.beta 3), did not support adhesion. A polyclonal antibody directed against both subunits of the VnR (alpha v, beta 3) inhibits attachment of 293 cells to vitronectin but not to other adhesive proteins. A beta 1-specific monoclonal inhibited attachment to fibronectin, laminin, and collagen IV, known ligands of beta 1 integrins, as well as vitronectin. This novel (alpha v. beta 1) VnR thus appears to mediate cell adhesion exclusively to vitronectin, in contrast to previously described VnRs which have multiple ligands.     

Assembly of type IV collagen. Insights from alpha3(IV) collagen-deficient mice

Type IV collagen includes six genetically distinct polypeptides named alpha1(IV) through alpha6(IV). These isoforms are speculated to organize themselves into unique networks providing mammalian basement membranes specificity and inequality. Recent studies using bovine and human glomerular and testis basement membranes have shown that unique networks of collagen comprising either alpha1 and alpha2 chains or alpha3, alpha4, and alpha5 chains can be identified. These studies have suggested that assembly of alpha5 chain into type IV collagen network is dependent on alpha3 expression where both chains are normally present in the tissue. In the present study, we show that in the lens and inner ear of normal mice, expression of alpha1, alpha2, alpha3, alpha4, and alpha5 chains of type IV collagen can be detected using alpha chain-specific antibodies. In the alpha3(IV) collagen-deficient mice, only the expression of alpha1, alpha2, and alpha5 chains of type IV collagen was detectable. The non-collagenous 1 domain of alpha5 chain was associated with alpha1 in the non-collagenous 1 domain hexamer structure, suggesting that network incorporation of alpha5 is possible in the absence of the alpha3 chain in these tissues. The present study proves that expression of alpha5 is not dependent on the expression of alpha3 chain in these tissues and that alpha5 chain can assemble into basement membranes in the absence of alpha3 chain. These findings support the notion that type IV collagen assembly may be regulated by tissue-specific factors.     

Quantitative analysis of collagen expression in embryonic chick chondrocytes having different developmental fates

A quantitative determination of collagen expression was carried out in cultured chondrocytes obtained from a tissue that undergoes endochondral bone replacement (ventral vertebra) and one that does not (caudal sterna). The "short chain" collagen, type X is only expressed in the former while the other "short chain" collagen type IX, was primarily expressed in the latter. These two tissues also differ in that vertebral chondrocytes express moderate levels of both type I procollagen mRNAs which were translated into full length procollagen chains both in vivo and in vitro, while caudal sternal chondrocytes did not. The percent of collagen synthesis was about 50% in both cell types, but sternal cells expressed twice as much collagen as vertebral cells even though type II procollagen was more efficiently processed to alpha-chains in vertebral chondrocytes than in sternal chondrocytes. The number of type II procollagen mRNA molecules/cell was found to be about 2300 in vertebral chondrocytes and about 8000 in sternal cells, in good agreement with the results reported by Kravis and Upholt (Kravis, D., and Upholt, W. B. (1985) Dev. Biol. 108, 164-172). There were about 630 copies of type I procollagen mRNAs with an alpha 1/alpha 2 ratio of 1.6 in vertebral chondrocytes compared with 5100 copies and an alpha 1/alpha 2 ratio of 2.2 in osteoblasts, and less than 40 copies in sternal cells. Since the rate of type I collagen chain synthesis was 50 times greater in osteoblasts than in vertebral cells, type I procollagen mRNAs were about six times less efficiently translated in vertebral cells than in osteoblasts. The type I mRNAs in vertebral chondrocytes were polyadenylated and had 5' ends that were identical in osteoblasts, fibroblasts, and myoblasts. Moreover, type I mRNAs isolated from vertebral chondrocytes were translated into full length preprocollagen chains in vitro in rabbit reticulocyte lysates. Thus, chondrocytes isolated from cartilage tissues with different developmental fates differed quantitatively and qualitatively in total collagen synthesis, procollagen processing, and distribution of collagen types.     

Molecular heterogeneity of basal laminae: isoforms of laminin and collagen IV at the neuromuscular junction and elsewhere

Laminin and collagen IV are components of most basal laminae (BLs). Recently, both have been shown to be products of multigene families. The A, B1, and B2 subunits of the laminin trimer are products of related genes, and the BL components merosin M and s-laminin are homologues of the A and B1 subunits, respectively. Similarly, five related collagen IV chains, alpha 1(IV)-alpha 5(IV), have been described. Here, we used a panel of subunit-specific antibodies to determine the distribution of the laminin and collagen IV isoforms in adult BLs. First, we compared synaptic and extrasynaptic portions of muscle fiber BL, in light of evidence that axonal and muscle membranes interact selectively with synaptic BL during neuromuscular regeneration. S-laminin, laminin A, and collagens alpha 3(IV) and alpha 4(IV) are greatly concentrated in synaptic BL; laminin B1 is apparently absent from synaptic BL; collagens alpha 1(IV) and alpha 2(IV) are less abundant in synaptic than extrasynaptic BL; and laminin B2 and merosin M are present at similar levels synaptically and extrasynaptically. These results reveal widespread differences between synaptic and extrasynaptic BL, and implicate several novel polypeptides as candidate mediators of neuromuscular interactions. Second, we widened our inquiry to assess the composition of several other BLs: endoneurial and perineurial BLs in intramuscular nerves, BLs associated with intramuscular vasculature, and glomerular and tubular BLs in kidney. Of eight BLs studied, at least seven have distinct compositions, and of the nine BL components tested, at least seven have distinct distributions. These results demonstrate a hitherto undescribed degree of heterogeneity among BLs.     

The alpha 5 chain of type IV collagen is the target of IgG autoantibodies in a novel autoimmune disease with subepidermal blisters and renal insufficiency

We describe a novel autoimmune disease characterized by severe subepidermal bullous eruptions and renal insufficiency with IgG autoantibodies directed against the NC1 domain of the alpha5(IV) collagen chain. In vivo deposits of IgG and C3 were found along the dermal-epidermal junction of skin lesions. The identity of the target antigen was determined by immunochemical analyses of candidate antigens using the patients' autoantibodies. The patients' IgG autoantibodies reacted with a 185-kDa polypeptide that was distinguished from the known autoantigens of the extracellular matrix including type XVII collagen, type VII collagen, or the alpha3, beta3, and gamma2 chains of laminin 5. Preincubation of the serum with recombinant alpha5(IV)NC1 domain of type IV collagen abolished immunoreactivity with the 185-kDa antigen. The serum reacted specifically with the alpha5(IV)NC1, among the six NC1 domains of type IV collagen, by Western blot and enzyme-linked immunosorbent assay analyses. The patients' autoantibodies reacted with normal skin and renal glomerulus but not with skin and glomerulus of a patient with Alport syndrome in which the basement membranes are devoid of the alpha5(IV) collagen chain. This study provided for the first time unambiguous evidence for the alpha5(IV) collagen chain as the target antigen in a novel autoimmune disease characterized by skin and renal involvement.     

Gene expression in visceral endoderm: a comparison of mutant and wild- type F9 embryonal carcinoma cell differentiation

We have examined the abundance and cell specificity of several mRNAs that are regulated during the retinoic acid (RA)-induced differentiation of F9 embryonal carcinoma cells to visceral endoderm. The experiments confirmed the multistep nature of this process by demonstrating the expression of the ERA-1/Hox 1.6 message within 6 h after RA addition; the expression of messages specific for the extracellular matrix proteins laminin B1 and B2, and collagen IV(alpha 1) between days 4 and 12; and the expression of two visceral endoderm markers, alpha-fetoprotein (AFP) and H19, by days 8-15. In situ hybridization experiments revealed that the collagen IV(alpha 1) mRNA is restricted to the outer cell layer of F9 cell aggregates regardless of the presence or absence of RA. Laminin B1 and B2 mRNAs are concentrated in the outer cell layer of RA-treated aggregates although significant levels of message are also observed within the interior cells of the aggregates. Unexpectedly, AFP mRNA is detectable in only a subset of the outer cells of F9 cell aggregates grown 15 d in the presence of RA. The results obtained from wild-type F9 cells were compared with those from a mutant F9 cell line, RA-5-1, which was previously shown to synthesize collagen IV containing six- to ninefold less 4-hydroxyproline than that in wild-type F9 cells. RA-5-1 cells exhibit four- to sixfold less of the mRNAs encoding two visceral endoderm proteins, AFP and H19, than wild-type F9 cells after RA treatment of RA-5-1 aggregates. RA-5-1 cells, however, do exhibit an RA-associated increase in the level of ERA-1/Hox 1.6 mRNA within 6 h after adding RA. Although the collagen IV protein level is similar in wild-type F9 and RA-5-1 aggregates, the collagen IV(alpha 1) message level is 6-20-fold greater in aggregates of mutant cells than in aggregates of wild-type cells. Moreover, in situ hybridizations showed that this message is evenly distributed throughout the RA-5-1 aggregates rather than restricted to the outer cell layers as it is in wild-type F9 aggregates. These results suggest that abnormal collagen IV expression and localization are associated with decreased expression of the visceral endoderm markers, AFP and H19, in RA-5-1 cell aggregates.     

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.     

Vitreous body collagen. Evidence for a dual origin from the neural retina and hyalocytes

Two different cells types have been shown to synthesize embryonic chick vitreous collagen (vitrosin) at different stages of development. Identification of vitrosin was established by labeling the embryos in ovo [3H]proline at stages 23 and 28 and separating the extracted vitreous collagen alpha-chains by carboxymethylcellulose chromatography. The labeled collagen consisted predominately of alpha 1 chains, indicating a molecule in the form of a trimer of identical chains designated (alpha 1)3. The molecular weight of the labeled chains measured approximately 95,000 daltons by molecular sieve chromatography, and contained 41% of their imino acid as 4- hydroxyproline. To establish which eye tissues synthesize vitrosine, the collagens produced in organ culture by the isolated neural retina, lens and vitreous body from stages 26-27, 29-30, and 40 were examined. At the two earlier stages, only the neural retina synthesized large quantities of (alpha 1)3 collagen whereas the lens and the cells within the vitreous body itself synthesized relatively small amounts of collagen characterized by an alpha 1:alpha 2 ratio of about 2:1. At stage 40, however, the cells of the vitreous body itself synthesized the greatest quantities of collagen, which now was predominantly an (alpha 1)3 type molecule. Stage 40 neural retina and lens synthesized lesser amounts of collagen with an alpha 1:alpha 2 ratio of 2 to 3:1. Chick vitrosin thus appears to be synthesized by the neural retina in early embryonic stages, whereas the major contribution derives from cells within the vitreous body in later development.     

Evidence for separate networks of classical and novel basement membrane collagen. Characterization of alpha 3(IV)-alport antigen heterodimer.

The COOH-terminal non-collagenous domains (NC1) of type IV collagen from glomerular basement membranes (GBM), lens capsule basement membranes, and Descemet's membrane varied in the distribution of their NC1 subunits. All of these basement membranes (BMs) contained both classical (alpha 1(IV) and alpha 2(IV)) and novel collagen chains (alpha 3(IV), alpha 4(IV) and the Alport antigen). Whereas GBM had a predominance of disulfide-bonded subunits, the lens capsule and Descemet's membrane were primarily monomeric, differences that are likely related to the functional and structural diversity of collagen in various tissues. A heterodimer formed from monomeric subunits of alpha 3(IV) and the Alport antigen exists in human and bovine GBM. This dimer represents an important cross-link of the NC1 domain of novel collagen. Additionally, immunoaffinity methodology showed that the novel BM collagen hexamers segregate into populations containing only novel BM subunits without the participation of the classical subunits (alpha 1(IV) and alpha 2(IV)). These data provided evidence for the presence of two separate networks of BM collagen: one containing alpha 1(IV) and alpha 2(IV), and the other consisting of the novel collagen chains.