Charles C. Clark

Biochemistry and Metabolism of Basement Membranes

Publisher Summary Basement membranes or basal laminae are ubiquitous in the vertebrate and invertebrate world and constitute important structural and functional extracellular matrices. Basement membranes are composed of fine fibrils, 40–60 A in diameter, arranged randomly in a granular matrix. Three general functions are ascribed to basement membranes: they (1) act as a semipermeable filter, (2) provide support, and (3) serve as a boundary between cell types or between cell layers and the underlying connective tissue. Basement membranes are insoluble in buffers at neutral pH, although small amounts of material can be brought into solution with acid buffers. Complete solubilization can be accomplished by reduction and alkylation in the presence of denaturant or after limited proteolysis. Collagen molecules are present in basement membranes. These apparently exist as triple-helical procollagen molecules composed of identical pro-α chains. Collagenous peptides equivalent in size to α chains can be isolated from digests of basement membranes. Noncollagenous glycoproteins are present in the matrix of most basement membranes; they are linked to the procollagen molecules via disulfide bonds and lysine- or hydroxylysine-derived cross-links.

Basement Membrane Zone Type XV Collagen Is a Disulfide-bonded Chondroitin Sulfate Proteoglycan in Human Tissues and Cultured Cells

Type XV collagen has a widespread distribution in human tissues, but a nearly restricted localization in basement membrane zones. The α1(XV) chain contains a highly interrupted collagenous region of 577 residues, and noncollagenous amino- and carboxyl-terminal domains of 530 and 256 residues, respectively. Cysteines are present in each domain and consensus sequences forO-linked glycosaminoglycans are situated in the amino terminus and in two large, noncollagenous interruptions. We now report that type XV collagen is a chondroitin sulfate proteoglycan in human tissues and cultured cells, and that the α chains are covalently linked by interchain disulfide bonds only between the two cysteines in the collagenous region. Western blotting of tissue extracts revealed a diffuse smear with a mean size ≥400 kDa, which after chondroitinase digestion resolved into a 250-kDa band in umbilical cord, and 250- and 225-kDa bands in placenta, lung, colon, and skeletal muscle. The latter two bands were also directly visualized by alcian blue/silver staining of a purified placenta extract. In a human rhabdomyosarcoma cell line, almost all of the newly synthesized type XV collagen was secreted into the medium and upon chondroitinase digestion just the 250-kDa α chain was generated. Chondroitinase plus collagenase digestion of tissue and medium proteins followed by Western blotting using domain-specific antibodies revealed a 135-kDa amino-terminal fragment containing glycosaminoglycan chains and a 27-kDa fragment representing the intact carboxyl terminus. However, a truncated carboxyl peptide of ∼8-kDa was also evident in tissue extracts containing the 225-kDa form. Our data suggest that the 225-kDa form arises from differential carboxyl cleavage of the 250-kDa form, and could explain the ∼19-kDa endostatin-related fragments (John, H., Preissner, K. T., Forssmann, W.-G., and Ständker, L. (1999)Biochemistry 38, 10217–10224), which may be liberated from the α1(XV) chain.

The embryonic rat parietal yolk sac: Changes in the morphology and composition of its basement membrane during development☆

Abstract The basement membrane (Reicherts membrane) of the entire capsular portion of the parietal yolk sac of rat embryos was examined both morphologically and chemically at various stages of gestation. The overall microscopic and compositional analyses showed Reicherts membrane to be typical of basement membranes isolated from other tissues and species. However, with increasing gestational age (from 11.5 to 17.5 days) a number of changes involving Reicherts membrane were noted: 1. The thickness increased rapidly then declined, while the surface area increased tenfold; 2. The total protein content increased twenty-fold while the collagen content increased eight-fold. As a result, the relative collagen content declined significantly; 3. The changes in the amino acid and carbohydrate composition were consistent with the latter finding. The observations listed above were evaluated in light of their possible relevance to an understanding of the morphogenesis of basement membranes during development, and to the possible mechanisms involved in pathogenesis of basement membrane dysfunction.

Up-regulation of Chondrocyte Matrix Genes and Products by Electric Fields

This study tested the hypothesis that selective and specific capacitively coupled electrical signals could stimulate gene expression and matrix production in bovine articular chondrocytes. Starting with a capacitively coupled electric signal that previously was shown to be effective in stimulating proliferation in bovine articular cartilage chondrocytes, dose responses were done sequentially for duration, response time, amplitude, duty cycle, and frequency. Results showed that a 0.5-hour, 20 mV/cm, signal at 60 kHz up-regulated aggrecan gene expression approximately eightfold (p < 0.0003) using a 50% duty cycle, whereas Type II collagen gene expression was up-regulated approximately fivefold (p < 0.02) using an 8.3% duty cycle. Using a compound signal (a 0.5-hour continuous period plus multiple 1-hour periods of 50% duty cycle for 7 days) both proteoglycan and collagen accumulation in vitro were increased approximately fivefold (p < 0.0003) and twofold (p < 0.0008), respectively. Also, the most effective capacitively coupled electric signal was different for each of the two molecules studied (aggrecan, 50% duty cycle and 4-hour response time; Type II collagen, 8.3% duty cycle and 6-hour response time). We conclude that selective up-regulation of gene expression and matrix accumulation of cartilage structural macromolecules (such as aggrecan and Type II collagen) with specific capacitively coupled fields occurs in vitro. This may be useful in vivo as a noninvasive modality to promote cartilage healing or ameliorate the effects of osteoarthritis, or both.

The Effect of Electrical Fields on Gene and Protein Expression in Human Osteoarthritic Cartilage Explants

BACKGROUND The destruction of cartilage in patients with osteoarthritis is a consequence of an imbalance between matrix synthesis and degradation. The purpose of the present study was to determine the effects of electrical stimulation on these processes in full-thickness osteoarthritic adult human articular cartilage explants. METHODS Full-thickness articular cartilage explants from osteoarthritic adult human knee joints were cultured in the absence or presence of interleukin-1beta (IL-1beta) and in the absence or presence of a specifically defined capacitively coupled electrical signal for seven or fourteen days. Total collagen and proteoglycan production were assessed by means of hydroxyproline and hexosamine analyses, respectively. Quantitative real-time polymerase chain reaction assays were used to measure mRNA expression levels of aggrecan, type-II collagen, collagenase-1 (MMP-1), collagenase-3 (MMP-13), stromelysin-1 (MMP-3), aggrecanase-1 (ADAM-TS4), and aggrecanase-2 (ADAM-TS5). RESULTS Electrical stimulation of cultured explants for seven or fourteen days resulted in significant increases (p < 0.007) in proteoglycan and collagen production and a highly significant upregulation (p <or= 0.005) of aggrecan and type-II collagen mRNA expression. This occurred even in the presence of IL-1beta. In the absence of IL-1beta, the expression of metalloproteinases was at barely detectable levels in these explants. Treatment with IL-1beta led to the significant upregulation of metalloproteinase expression (p < 0.03), but simultaneous administration of the capacitively coupled electrical signal dramatically inhibited this stimulation. CONCLUSIONS The data show that, even in the presence of IL-1beta, a specific, defined capacitively coupled electrical signal can result in significant upregulation of cartilage matrix protein expression and production while simultaneously significantly attenuating the upregulation of metalloproteinase expression. These results support the contention that delivery of a specific, defined electrical field to articular cartilage could result in matrix preservation.

In vitro bone-cell response to a capacitively coupled electrical field. The role of field strength, pulse pattern, and duty cycle.

Newborn rat calvarial bone cells were grown to confluence and subjected to a matrix of sine wave 60-kHz capacitively coupled electrical signals of various field strengths, pulse-burst patterns, and duty cycles. Both [3H] thymidine incorporation into DNA and alkaline phosphatase activity were evaluated in field strengths ranging from 0.0001 to 20 mV/cm, with pulse-burst patterns ranging from continuous to 5 milliseconds ON/495 milliseconds OFF, with daily duty cycles ranging from 0.25% to 25%. A significant increase in proliferation occurred in field strengths of 0.1, 1, and 20 mV/cm when the signal was applied continuously for six hours. Significant proliferation also occurred when the 20-mV/cm field was pulsed for six hours at 5 milliseconds ON/495 milliseconds OFF and at 5 milliseconds ON/245 milliseconds OFF. No change in alkaline phosphatase activity occurred in the 20-mV/cm field with any signal. At 1 mV/cm, there was a significant decrease in alkaline phosphatase activity in the continuous signal and in the 5 milliseconds ON/62 milliseconds OFF signal; in the lower fields evaluated, there was an actual decrease in alkaline phosphatase activity with some of the signals. The field strength plays a dominant role in determining the bone-cells proliferative response, and to a lesser extent the alkaline phosphatase activity response, to a capacitively coupled electric field. The pulse configuration and the duty cycle are also important, but only if the proper field strength is being applied to the cell.

Signal transduction in electrically stimulated articular chondrocytes involves translocation of extracellular calcium through voltage-gated channels

OBJECTIVE To ascertain, using specific inhibitors, the potential role of calcium-related signal transduction pathways in the mechanism of cartilage matrix protein gene induction and metalloproteinase gene suppression by capacitively coupled electric fields. METHODS Articular chondrocytes were isolated from adult bovine patellae and cultured in high density for 7 days. To study matrix protein expression, cells cultured in the absence or presence of specific calcium pathway inhibitors were exposed to a capacitively coupled electrical field (60 kHz, 20 mV/cm): for aggrecan 1h at 50% duty cycle and for type II collagen 6h at 8.3% duty cycle. To study metalloproteinase expression in the presence of interleukin 1 beta (IL-1beta), cells were cultured as above but exposed for only 30 min to a 100% duty cycle signal. At harvest, total mRNA was isolated and aggrecan, type II collagen, matrix metalloproteinase (MMP-1, -3 and -13) and aggrecanase [a disintegrin and metalloproteinase with thrombospondin repeats (ADAMTS-4 and -5)] mRNA expression were measured by quantitative real-time polymerase chain reaction (qPCR). RESULTS (1) In the absence of inhibitors, appropriate electrical stimulation induces a 3-4-fold up-regulation of both aggrecan and type II collagen mRNA and a 3.7-9.6-fold down-regulation of IL-1beta-induced metalloproteinases; (2) the presence of inhibitors alone does not affect any target mRNA levels; (3) inhibitors of intracellular calcium regulation and inositol 1,4,5-triphosphate (IP(3)) formation [8-(diethylamino)octyl-3,4,5,-trimethoxybenzoate hydrochloride (TMB-8) and neomycin, respectively] have no effect on regulation of target mRNA levels by electrical stimulation; and (4) inhibitors of voltage-gated calcium channels (verapamil), calmodulin activation (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride, W-7), calcineurin activity (cyclosporin A), phospholipase C activity (bromophenacyl bromide, BPB) and prostaglandin E(2) (PGE(2)) synthesis (indomethacin) completely inhibit the effects of electrical stimulation. CONCLUSIONS The results are consistent with the effects of electrical stimulation involving a pathway of extracellular Ca(2+) influx via voltage-gated calcium channels rather than from intracellular Ca(2+) repositories; and with downstream roles for calmodulin, calcineurin and nuclear factor of activated T-cells (NF-AT) rather than for phospholipase C and IP(3).

Isolation and partial characterization of precursors to minor cartilage collagens.

Suspension cultures of cartilage cells were prepared from 17-day chick embryo sterna and radiolabeled with [14C]-proline under conditions which sought to minimize proteolytic conversion of procollagen to collagen. Collagenous proteins were isolated from the culture medium and cell fraction, were purified in their native state by (NH4)2SO4 precipitation and DEAE-cellulose chromatography, and were characterized by protease susceptibility, SDS-gel-filtration and SDS-polyacrylamide gel electrophoresis. Qualitatively, the precursor components present in the medium were similar to those in the cell extract; quantitatively, it appeared that the minor cartilage collagen precursor components derived from 1 alpha, 2 alpha, 3 alpha and type IX collagens were more prevalent in the cell extract. SDS-PAGE of unreduced samples showed that precursors to both of these collagens migrated as distinct high-molecular-weight aggregates. After chymotrypsin digestion, unreduced type IX collagen migrated as two disulfide-bonded aggregates--a large one (Mr approximately 210K) and a small one (Mr approximately 43K); whereas 1 alpha, 2 alpha, 3 alpha chains migrated identically whether reduced or unreduced. Reduction of undigested type IX aggregate yielded two components of Mr approximately 97K and 78K; whereas reduction of the chymotrypsin resistant 210K and 43 K aggregates gave a single component of Mr approximately 61K and a component which migrated at the dye front, respectively. The molecular origin of these components was confirmed by differential NaCl precipitation. It was concluded that this culture system synthesized precursors to 1 alpha, 2 alpha, 3 alpha and type IX collagens in addition to type II; type X collagen was not detected even though the 17-day sternum contained a population of cells morphologically similar to hypertrophic chondrocytes. The precursor chains to 1 alpha, 2 alpha, 3 alpha collagen had an apparent Mr greater than pro-alpha (II) and could be isolated as a disulfide-bonded aggregate(s); the precursor chains to type IX collagen had an apparent Mr less than pro alpha (II) and could also be isolated as a disulfide-bonded aggregate. All of the cartilage collagen precursors had protease-susceptible regions, but those in type IX appeared to be more sensitive to pepsin than to chymotrypsin.

Chondroitin sulfate proteoglycan is a constituent of the basement membrane in the rat embryo parietal yolk sac

SummaryIn addition to containing Type IV collagen, laminin and entactin, basement membranes contain small amounts of proteoglycans substituted primarily with heparan sulfate chains. We have previously shown, however, that parietal yolk sacs in organ culture synthesize predominantly chondroitin sulfate proteoglycan. In the present study, we have used histochemical and immunohistochemical techniques coupled with chondroitinase ABC digestion to provide evidence for the presence of chondroitin sulfate proteoglycan in the basement membrane (Reicherts membrane) of the 14.5-day rat embryo parietal yolk sac. The results revealed numerous cuprolinic blue-positive filaments and granules, 20–30 nm in greater length or diameter, dispersed throughout the thickness of the basement membrane. Both structures were removed by preincubating freshly isolated parietal yolk sacs with chondroitinase ABC. A similar labeling pattern was also obtained with immunoelectron microscopy using gold-labeled monoclonal anti-bodies directed against the three major isomers of protein-bound chondroitin sulfate. In contrast, coarser cuprolinic blue granules, 40–100 nm in diameter, were neither sensitive to chondroitinase ABC digestion nor labeled by the monoclonal antibodies. These results thus indicate that Reicherts membrane contains chondroitin sulfate proteoglycan in addition to heparan sulfate proteoglycan.

Up‐regulation of expression of selected genes in human bone cells with specific capacitively coupled electric fields

The objective of the described experiments was to determine the electrical parameters that lead to optimal expression of a number of bone‐related genes in cultured human bone cells exposed to a capacitively coupled electric field. Human calvarial osteoblasts were grown in modified plastic Cooper dishes in which the cells could be exposed to various capacitively coupled electric fields. The optimal duration of stimulation and optimal duration of response to the electrical field, and the optimal amplitude, frequency and duty cycle were all determined for each of the genes analyzed. Results indicated that a capacitively coupled electric field of 60 kHz, 20 mV/cm, 50% duty cycle for 2 h duration per day significantly up‐regulated mRNA expression of a number of transforming growth factor (TGF)‐β family genes (bone morphogenetic proteins (BMP)‐2 and ‐4, TGF‐β1, ‐ β2 and ‐β3) as well as fibroblast growth factor (FGF)‐2, osteocalcin (BGP) and alkaline phosphatase (ALP). Protein levels of BMP‐2 and ‐4, and TGF‐β1 and ‐ β2 were also elevated. The clinical relevance of these findings in the context of a noninvasive treatment modality for delayed union and nonunion fracture healing is discussed.