Michael A. Haralson

Mesangial deposition of type I collagen in human glomerulosclerosis

The presence of type I collagen in both diffuse and nodular diabetic glomerular lesions has been examined using immunohistochemical and electron microscopic techniques. At the ultrastructural level, banded collagen fibrils were observed in the mesangium in all cases of nodular (Kimmelstiel-Wilson) sclerosis and in 60% of the diffuse sclerotic lesions. Antibodies against type I collagen were localized in the fibrotic interstitium and the mesangium in all cases examined. Staining with type I collagen antibodies occurred in glomeruli with intact Bowmans capsules, and was predominantly localized to areas immediately adjacent to mesangial cells. In cases of focal sclerosis of nondiabetic origin, banded collagen fibrils and staining with anti-type I collagen antibody were observed in all cases in which the segmental lesion was presented in the specimen. The pattern of antibody localization in both the diabetic lesions and focal sclerosis differed from that obtained using anti-type IV (basement membrane) collagen antibodies. These results demonstrate that type I collagen is among the extracellular matrix components that comprise the sclerotic glomerular lesions of both diabetic and nondiabetic origin. Furthermore, the spatial localization of this collagen type suggests mesangial cell origin.

Synthesis of Type III Collagen by Cultured Kidney Epithelial Cells

Studies have been performed to evaluate both the relative amounts and molecular forms of the collagens synthesized by an established line of cultured rat kidney epithelial (clone NRK52E) cells. The collagens secreted into the culture medium and extracted from the cell layers of cultured NRK52E cells were isolated after limited pepsin digestion and differential salt fractionation. Greater than 95% of the collagenous proteins synthesized by NRK52E cells were found to be associated with the cells and not secreted. Polyacrylamide gel electrophoresis under denaturing conditions of the NRK52E cell collagens indicated the presence of components exhibiting properties corresponding to those of the chains present in types I, III, IV and V collagen. Analysis of each fraction by carboxymethyl-trisacryl chromatography revealed that approximately two-thirds of the total collagen synthesized by NRK52E cells was type III. Of the remaining collagen types I, IV and V molecules represented 20%, 4% and 10% respectively, of the total produced. Essentially all of the type I collagen produced by NRK52E cells was recovered as the type I-trimer, whereas the type V molecules synthesized by NRK52E cells had the molecular compositions of [alpha 1(V)]2 alpha 2(V) and alpha 1(V)alpha 2(V)alpha 3(V). These data establish the relative proportions and molecular forms of the collagens synthesized by cultured NRK52E cells. Furthermore, these findings suggest that NRK52E cells may be a useful in vitro model for investigating the regulation of changes in collagen biosynthesis occurring under situations of renal epithelial cell injury.

Evidence that the collagen in the culture medium of Chinese hamster lung cells contains components related at the primary structural level to the α1(V) collagen chain

The collagenous protein synthesized by cultured Chinese hamster lung (CHL) cells and present in the culture medium has been isolated after limited pepsin digestion and differential salt precipitation. Molecular size analysis of this material indicates that the CHL cell medium collagen contains chains which exhibit an apparent molecular mass of approximately 85,000 Da. When chromatographed on CM-cellulose under denaturing conditions, the reduced and alkylated CHL cell medium collagen chains elute slightly after the human alpha1(I) chain but well before the pepsin-derived alpha1(V) chain, which is the constituent chain present in the CHL cell cellular matrix collagen. Analysis of the peptides derived by CNBr cleavage of the CHL medium collagen chains by chromatography on CM-cellulose reveals, however, that these chains contain peptides which correspond both in size and in chemical properties to those derived from the alpha1(V) collagen chain, but clearly lack two peptides (alpha1(V)-CB4 and alpha1(V)-CB5) which are normally present in pepsin-derived alpha1(V) chains. Furthermore, analysis of the CHL cell culture medium collagenous material obtained without pepsin digestion indicates the presence of collagenous chains that exhibit after reduction a molecular mass of approximately 160,000 Da, which is smaller than the proposed size of the pro alpha1(V) collagen chain. These results demonstrate that the collagenous protein present in the culture medium of CHL cells is directly related at the primary structural level to the alpha1(V) collagen chain, and it is postulated that this material represents the large fragment derived from a collagenase cleavage of the [pro alpha1(V)]3 molecules present in the cell layer. Furthermore, these results and previous reports indicate that the only identifiable genetic type of procollagen chain synthesized by this cloned cell line in culture corresponds to the pro alpha1(V) chain.

Characterization of the Collagen Phenotype of Rabbit Proximal Tubule Cells in Culture

Studies were performed to characterize the collagen phenotype of cultured rabbit proximal tubule (RPT) epithelial cells grown on plastic and on the reconstituted basement membrane preparation, Matrigel. When grown on a plastic substratum, RPT cells display a cobblestone appearance characteristic of glomerular epithelial cells. While initially forming an interlocking network of cells after subculture on Matrigel, this pattern of culture morphology rapidly develops into one characterized by isolated, organized groups of cells. Notwithstanding the effects of Matrigel on culture morphology, total cellular proliferation was reduced only 25% when RPT cells were grown on this substrate. Greater than 90% of the collagen synthesized by RPT cells grown on plastic was secreted into the culture medium. Qualitative analysis by SDS-PAGE revealed components exhibiting electrophoretic mobilities corresponding to the chains present in type IV and type I collagens. Quantitative analysis by CM-Trisacryl chromatography established that approximately 2/3 of the total collagen synthesized by RPT cells grown on plastic was type IV and approximately 1/3 type I. Quantitative analysis of the collagens produced by RPT cells grown on Matrigel again indicated the synthesis of only type IV and type I molecules but in a slightly more equal ratio of both collagen types and in the ratio of secreted to cell-associated molecules. However, the total amount of collagen synthesized by RPT cells grown on Matrigel was reduced to approximately 1% of the level synthesized by the cells grown on plastic. On plastic, approximately 3/4 of the type I collagen produced was recovered as the type I homotrimer, but on Matrigel type I homotrimers represented only approximately 55% of the total type I collagen synthesized. On Matrigel, the majority of the type IV collagen was recovered as heterotrimers containing alpha1(IV) and alpha2(IV) chains. In contrast, RTP cells grown on plastic predominantly produced type IV homotrimers containing only the alpha1(IV) chain. These data represent the initial report describing the collagens produced by nonimmortalized cultured proximal tubule cells. The finding that a significant amount of the total collagen synthesized was type IV (basement membrane) collagen, regardless of culture substrate, suggests that the RPT cells have maintained a significant degree of differentiation in culture, and thus establishes RPT cells as an appropriate model for investigating ECM changes in proximal tubule cells that occur in kidney disease. Finally, the observation that culture of RPT cells on a reconstituted basement membrane preparation results in a significant reduction in total collagen production and alterations in the molecular forms of type IV and type I molecules synthesized indicates that integrity of the tubular basement membrane may represent an important component in preventing the development of tubulointerstitial fibrosis.

The Effects of Tissue Pretreatment and Pepsin Levels on the Isolation of Collagens from Human Placenta

A comparative study has been undertaken to ascertain the effects of different tissue pretreatment procedures on the recovery of the major genetic types of collagen from human placenta. Essentially the same recovery of types I, III, IV and V collagen was obtained from placenta which was directly processed, from placenta which was stored at -70 degrees C after washing, and from dried acetone extracts of this tissue. Each collagen type isolated from the treated tissue preparations displayed properties consistent with those exhibited by its counterpart obtained from fresh tissue which was directly processed. Furthermore, it was observed that while the amount of types I and III collagen recovered was directly proportional to the level of pepsin employed, the recovery of types IV and V collagen was inversely related to this isolation parameter. These results establish that human placenta can be either stored frozen or as a dry acetone extract without affecting either the yield or the profile of the different genetic types of collagen recovered and that the amounts of different genetic types of collagen recovered can be modulated by the amount of pepsin employed in the isolation.

Cell-free synthesis of Chironomus thummi (Diptera) globins

Abstract RNA isolated from Chironomus thummi (Diptera) larvae directs the incorporation of amino acid into newly synthesized products in a cell-free translation system prepared from wheat germ. A fraction of the total cell-free product was specifically immunoprecipitable with antibody against total C. thummi hemoglobin. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis of the immunoreactive material revealed the cell-free product to have an apparent molecular mass approximately 3000 daltons greater than secreted C. thummi globin purified from hemolymph. In contrast, analysis of the immunoreactive material by polyacrylamide gel electrophoresis under nondenaturing conditions indicated several chemically distinct globins to be present in the cell-free immunoreactive products. These results provide evidence suggesting the possible existence of a preglobin and the data further provide the initial foundation required for elucidating the regulatory mechanisms that control the developmental stage-specific expression of the globin genes in C. thummi .

Effects of epidermal growth factor on collagen expression by rat kidney mesangial cells in culture

Increased collagen production by mesangial cells plays a key role in the development and progression of glomerular sclerosis. These changes reflect in part the impact of growth factors on mesangial cells. Since mesangial cells possess receptors for epidermal growth factor (EGF) and since previous studies have documented that EGF affects collagen synthesis in other cell types, we have examined the effects of EGF on collagen biosynthesis by rat kidney mesangial (RKM) cells in culture. Exposure for 24 h to EGF did not substantially affect the growth rate of RKM cells. While the types of collagen produced by RKM cells (types I, III, IV and V) were unaltered by exposure to EGF, total collagen production was reduced ( approximately 50%). This decrease in collagen expression was not uniform for each collagen type. Type I collagen production was inhibited by approximately 50%, both type III and type IV expression were each reduced by approximately 30%, but type V collagen production was suppressed by only approximately 15%. The reduction in type I collagen synthesis was accounted for mainly by a decrease in type I homotrimer production. Since type I molecules represent approximately 95% of the total collagen produced, the decrease in overall collagen expression reflects a specific suppression by EGF on type I homotrimer production in mesangial cells. As EGF exposure resulted in a decrease in collagen production, these results suggest that the increases in synthesis and deposition of collagen observed in several glomerular diseases likely do not reflect the short-term effects of EGF on mesangial cells. Rather, these findings suggest the possibility that EGF or EGF-like growth factors may ameliorate the effects of other soluble factors that cause enhanced matrix production and deposition in renal diseases.

The extracellular matrix in large-cell lymphomas: a histopathologic, immunohistochemical, and ultrastructural study

We studied the histopathologic, immunohistochemical, and ultrastructural features of the extracellular matrix (ECM) in 20 immunologically defined large-cell lymphomas, including immunoblastic sarcoma of B cells (three patients), peripheral T cell lymphoma (five patients), large non-cleaved follicular center cell (FCC) lymphoma (nine patients), and non-marking large-cell lymphoma (three patients). Immunohistochemical studies were performed with antibodies to laminin, fibronectin, and collagen types I, III, IV, and V. The immunologically defined subgroups demonstrated characteristic differences in ECM in light microscopic appearance, composition, and ultrastructural features. Immunoblastic sarcomas of B cells showed delicate intercellular bands that were apparent only at high power but were distinct in exhibiting focal staining for basement membrane elements (laminin and type IV collagen) in addition to type I collagen and fibronectin. Electron microscopically, no basal lamina were apparent, although the collagen fibers were embedded in a dense matrix not seen in the other lymphomas. All peripheral T cell lymphomas exhibited a packeting pattern of intercellular bands and were distinguished by the frequent presence of intense pericellular staining for type V collagen as well as by focal pericellular staining for types I and III collagen. The latter finding corresponded to extensive areas of direct contact between tumor cells and the ECM by electron microscopy. The large non-cleaved FCC lymphomas and the non-marking large-cell lymphomas demonstrated both diffuse and compartmentalizing arrangements of intercellular bands that frequently coexisted and stained predominantly for fibronectin and types I and III collagen. All groups demonstrated myofibroblasts and fibroblasts partially or completely separating the ECM from tumor cells, suggesting that most of the ECM is part of a reaction to these lymphomas. These studies show more variation in light microscopic appearance, composition, and ultrastructural relationships of the intercellular and pericellular ECM than was apparent in earlier studies of cleaved FCC lymphomas.

Cell-free synthesis of putative type V procollagen chains programmed by Chinese hamster lung cell mRNA.

A messenger RNA fraction isolated from cultured Chinese hamster lung (CHL) cells programs in a cell-free system prepared from wheat germ the efficient incorporation of [14C] proline into newly synthesized protein with a significant fraction of the incorporated substrate being digestible with bacterial collagenase. This reaction requires both subcellular fractions, an energy source, and is inhibited by the antibiotic puromycin. The relative amount of collagenase-digestible to non-digestible cell-free product depends upon the ratio of CHL mRNA to wheat germ lysate, is not affected by either the Mg2+ or K+ concentrations employed, and under optimal condition, approximately 38% of the total incorporated substrate is collagenase-sensitive. Electrophoresis on SDS-polyacrylamide gels of the products programmed by CHL mRNA indicates that the collagenase-digestible material corresponds in size to a procollagen chain with an apparent molecular mass of approximately 170,000 daltons. These studies suggest that the collagen alpha 1 (V) chain is initially synthesized as a precursor procollagen chain and demonstrate that a significant amount of the mRNA in Chinese hamster lung cells codes for this protein.