Alterations in the basement membrane (heparan sulfate) proteoglycan in diabetic mice.

We have grown the EHS (Engelbreth-Holm, Swarm) tumor in normal and genetically diabetic mice (db/db) and measured some components of basement membrane produced in the tumor. These studies showed similar amounts of total protein in control and diabetic tissue and similar patterns of proteins on SDS gel electrophoresis of extracts of the tissue. Laminin, a basement membrane specific glycoprotein utilized as an attachment factor by epithelial cells, was present in increased amounts in diabetic tissue. In contrast, the amount of BM-1 (heparan sulfate) proteoglycan was reduced. Less 35S-sulfate was incorporated into this proteoglycan, and the proteoglycan, but not its component glycosaminoglycans, was heterogeneous in size. The data indicate that either the synthesis of proteoglycan was decreased or its degradation was increased in diabetic tissue. Since the heparan sulfate proteoglycan serves to block the passage of anionic macromolecules through the basement membrane, decreased levels could account for the increased porosity of diabetic basement membrane. Compensatory synthesis of the basement membrane components to restore normal permeability could account for the thickened basement membranes observed in diabetes.

Response of diabetic basement membrane--producing cells to glucose and insulin.

The effect of insulin and glucose on the synthesis of basement membrane components was studied in organ cultures of a basement membrane-producing tumor grown in diabetic and normal mice. Tumor tissue grown in diabetic mice produced more protein and basement membrane-specific proteoglycan in response to insulin than tissue grown in normal mice. Addition of high levels of glucose to the culture medium did not alter insulin-stimulated protein synthesis by diabetic or normal tissue but dampened insulin-stimulated production of proteoglycan. These data suggest that basement membrane-producing cells in diabetic hosts may be hypersensitive to insulin and that stimulation of protein production by insulin may play some role in the in situ hypertrophy of basement membranes.

Gene mapping of human ocular connective tissue proteins. Assignment of the structural gene for corneal type I procollagen to human chromosome 7 in human corneal stroma-mouse fibroblast somatic cell hybrids.

Somatic cell hybrids between mouse and human corneal stroma fibroblasts have been used to identify the human chromosome responsible for the synthesis of human corneal type I procollagen. Twenty-six separate hybrid clones and subclones from three separate hybridization events were isolated in hypoxanthine-aminopterin-thymidine (HAT) selection medium and were used to assay for the production of human type I procollagen. Human and mouse chromosomes were identified in each hybrid clone by alkaline Giemsa staining of metaphase chromosomes spread and by isozyme analysis. We have found that human type I procollagen production segregates concordantly with human chromosome 7 and with beta-glucuronidase, another human chromosome 7 marker. The pro-alpha 1 gene and possibly the pro-alpha 2 gene appear to be encoded on human chromosome 7. Because we have previously assigned the gene for human skin type I procollagen to chromosome 17, our present data indicate that separate genes and control mechanisms must exist for skin and corneal type I procollagen.

In vitro production of basement membrane collagen by a clonal line of mouse lens epithelial cells.

The basement membrane is a structure of prime importance for the proper functioning of certain organs such as the lens and the kidney. Its inaccessibility and resistance to extraction, however, make an absolute determination of its composition difficult. The establishment of a cell line that synthesizes authentic basement membrane components in vitro would make basement membrane components more easily obtained, and would provide a controlled situation which could be more easily manipulated. In this report, a Balb/C mouse lens epithelial cell line was investigated. The cells were observed to undergo morphological transformations in vitro depending upon the cell density. As cell-cell contacts became prevalent, stellate cells organized into an epithelioid sheet. Later, elongate cells and lentoid bodies predominated in the culture. Thus, morphologically the cells mimicked the in vivo transition of the lens epithelial cells into lens fiber cells. Furthermore, the collagen(s) synthesized by these cells reacted specifically with affinity purified antibody directed against mouse type IV collagen. These morphological and immunological data lend credence to the concept that this lens epithelial cell line is an authentic replica of the in vivo situation.

Reduced aminergic synthesis in the hypothalamus of the infertile, genetically diabetic (C57BL/KsJ-db/db) male mouse.

Diabetes mellitus is commonly associated with reproductive neuroendocrinopathy in both humans and animal models for the disease. Diabetes-associated reproductive failure in the male is a result of multilevel dysfunction within the hypothalamo-pituitary-testicular axis. In view of the known effects of diabetes on hypothalamic gonadotropin-releasing hormone (GnRH) and gonadotropins in chemically-induced animal models for diabetes, we examined hypothalamic aminergic activities (important to the regulation of GnRH release), circulating gonadotropin levels and testicular morphology in the infertile, genetically diabetic (C57BL/KsJ-db/db) male mouse. Groups of 2-5 month old (average age: 3.4 months) and 6-11 month old (average age: 8.8 months) diabetic mice were compared with age-matched non-diabetic (C57BL/KsL(-)+/?) male mice. Diabetic mice in both age groups were markedly obese and hyperglycemic. Hypothalamic serotonin synthesis was inhibited in the preoptic area-anterior hypothalamus (POA-AH) in both 2-5 month old and 6-11 month old diabetic mice as well as in the mediobasal hypothalamus-median eminence (MBH-ME) of 6-11 month old diabetic mice. Catecholamine synthesis (norepinephrine and dopamine) was reduced in the POA-AH of 2-5 month old diabetic mice and in the MBH-ME of 6-11 month old mice. These aminergic changes were associated in 2-5 month old diabetic mice with reduced circulating levels of LH and in 6-11 month old diabetic mice, of both LH and FSH. In 6-11 month old diabetic mice, testes were characterized by a thickened tunica albuginea, numerous Sertoli cells and the near absence of any spermatogenic cells. The epididymis from these diabetic mice was devoid of spermatozoa.(ABSTRACT TRUNCATED AT 250 WORDS)

Loss of the basement membrane matrix molecule, bamin, in diphenylamine-treated mice.

Polycystic kidney disease (PKD) is a life-threatening disease characterized by focal dilatations or cysts in certain kidney tubules. Changes (i.e. thickening) in the support structure for these tubules, the basement membrane, have been related to the development of the cysts. Analysis of changes in basement membranes of humans with PKD is difficult, however, due to the restricted amount of material available for study. Several genetic and induced animal models, including diphenylamine-treated rats, have been employed to study the effects of PKD on basement membrane synthesis. While all these studies agree that PKD has a significant influence on basement membranes, no clear understanding as to how PKD effects basement membrane composition has emerged. Here, we report our findings of the effect of diphenylamine treatment on the composition of the basement membrane. Our immunohistological studies indicate that bamin, a recently described glycoprotein associated with glomerular basement membranes (Robinson et al., 1989), is not present in the glomerular basement membranes of diphenylamine-treated mice. This finding was confirmed by analysis of the composition of the basement membrane matrix synthesized by EHS tumors grown in control and diphenylamine-treated mice. The possible role of bamin in the pathogenesis of renal cysts is discussed.

Role of laminin in the attachment of PAM 212 (epithelial) cells to basement membrane collagen.

Measurements of the attachment of the PAM 212 line of mouse epithelial cells to various collagen substrates show that these cells adhere preferentially to type IV, basement membrane collagen. Neither serum nor fibronectin stimulated the attachment of these cells (unlike fibroblasts) to type IV collagen. Preincubation of the PAM 212 cells with cycloheximide prevented attachment. Thus these cells do not attach by means of a macromolecule present inserum, but instead synthesize an attachment factor. Extracts of the EHS tumor, which produces an extracellular matrix containing basement membrane components, were tested for their ability to promote attachment to cycloheximide-treated PAM 212 cells. Saline extracts of the tumor stimulated the attachment of the PAM 212 cells to type IV collagen in the presence of cycloheximide. Laminin, a high molecular weight glycoprotein constituent of basement membrane, was purified from the salt extract and was found to be the active species at concentrations as low as 1-5 microgram/ml. When laminin was preincubated on plates coated with either type I, II, III, IV, or V collagen and the plates subsequently washed, high levels of attachment were seen only on type IV collagen-coated plates. Affinity purified antibody directed against laminin inhibited the attachment of PAM 212 cells to a type IV collagen substrate. Laminin appears to be a specific attachment protein for epithelial cells since it did not stimulate the attachment of fibroblasts to type I or to type IV collagen substrates. These data suggest that lamin is produced and utilized by these epithelial cells to attach to basement membrane collagen.

Characterization of a novel glycoprotein isolated from the basement membrane matrix of the Engelbreth-Holm-Swarm tumor.

A previously undescribed protein has been isolated and purified from the extracellular matrix of the Engelbreth-Holm-Swarm (EHS) tumor, a murine tumor that synthesizes an extensive matrix composed of basement membrane molecules. Molecular characterization of the molecule determined that it is a glycoprotein with internal disulfide bonds and an isoelectric point of 6.0. Electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the glycoprotein migrated as a diffuse band with a molecular weight of approximately 72,000-80,000. The amino acid composition was significantly different from known basement membrane components. Polyclonal antibodies that specifically recognize the glycoprotein localized it to the kidney glomerular basement membrane. These antibodies did not cross-react with either known basement membrane components (laminin, type IV collagen, and heparan sulfate proteoglycan), with 70K "culture shock" protein or with components of normal mouse serum (including mouse transferrin, albumin, or alpha-fetoprotein), when analyzed by "Western" immunoblots. Our data indicate that the glycoprotein is synthesized by the EHS tumor cells and is present at relatively high levels in the EHS tumor matrix.

The adhesive glycoprotein laminin is an agglutinin.

The glycoprotein laminin appears to function in the attachment of various epithelial cells to basement membranes. We examined whether its putative cell-adhesive activity could be analyzed in a simple, one-component model system--the agglutination of erythrocytes. Laminin is a potent agglutinin of aldehyde-fixed sheep and human erythrocytes, with half-maximal agglutination at 0.8 micrograms/ml in a standard hemagglutination assay. Inhibitors of this hemagglutinating activity include gangliosides and certain charged phospholipids. The spectrum of molecules is similar but not identical to inhibitors of the hemagglutinating activity of the adhesive glycoprotein fibronectin. Laminin is much less biologically active in three other assays for fibronectin biological activity involving cell spreading on tissue culture substrates, attachment of fibroblastic cells to type I collagen, and restoration of normal morphology to transformed fibroblasts. The adhesive glycoproteins laminin and fibronectin therefore differ markedly in biological activities in several specific adhesion assays; however, they resemble one another in binding to heparin, collagen, and cell surfaces and in their agglutinin activity.

Structure, function, and pathology of basement membranes.

Basement membranes support epithelial and endothelial cells, prevent the passage of proteins, and generate histologically distinct compartments in the body. Basement membranes contain a number of components, only some of which have been isolated and characterized. These include type IV collagen, heparan sulfate proteoglycan, laminin, entactin, and fibronectin. Some components, such as bullous pemphigoid antigen and Goodpasture antigen, are present only in specific tissues, such as the skin or the kidneys. Alterations in basement membranes are associated with various diseases. For example, metastatic cells are able to attach to basement membranes and to degrade them. Such interactions with basement membranes underlie the ability of these cells to penetrate tissues and to spread in the body. In diabetes, basement membranes are thickened but are more porous, which is possibly due to a decreased amount of heparan sulfate proteoglycan. Basement membranes are also the site of immunopathologic disorders.

Reduced synthesis of basement membrane heparan sulfate proteoglycan in streptozotocin-induced diabetic mice.

In diabetes, certain basement membranes become thicker yet more porous than normal. To identify possible changes in the basement membrane, we have grown the Engelbreth-Holm-Swarm tumor, a tissue that produces quantities of basement membrane in normal mice and in streptozotocin-treated, insulin-deficient, diabetic mice. The level of laminin, a basement membrane-specific glycoprotein, and the level of total protein were slightly elevated in the diabetic tissue. In contrast, the level of the basement membrane specific heparan sulfate proteoglycan was only 20% of control. The synthesis of this proteoglycan was also reduced in the diabetic animals, while the synthesis of other proteoglycans by tissues such as cartilage was normal. The synthesis of the heparan sulfate proteoglycan in diabetic animals was inversely related to plasma glucose levels showing an abrupt decrease above the normal range of plasma glucose. Insulin restored synthesis to normal but this required doses of insulin that maintained plasma glucose at normal levels for several hours. Since the heparan sulfate proteoglycan in the basement membrane restricts passage of proteins, its absence could account for the increased porosity of basement membrane in diabetes. A compensatory synthesis of other components could lead to their increased deposition and the accumulation of basement membrane in diabetes.

Localization of the human fibronectin (FN) gene on chromosome 8 by a specific enzyme immunoassay.

The fibronectin produced by clonal murine-human hybrid cell lines containing various complements of human chromosomes was measured. Human and murine fibronectins were assayed by specific immunoassay, and the production of human fibronectin was correlated with karyology and isozyme markers for specific human chromosomes. The data show a 100% concordance between the expression of human fibronectin and glutathione reductase, a marker for human chromosome 8, indicating that chromosome 8 codes for the fibronectin polypeptide.