Connective tissue growth factor (CTGF) expression is regulated by transforming growth factor-␤ (TGF-␤) and strong up-regulation occurs during wound healing; in situ hybridization data indicate that there are high levels of CTGF expression in fibrotic lesions. Recently the binding parameters of CTGF to both high and lower affinity cell surface binding components have been characterized. Affinity cross-linking and SDS-polyacrylamide gel electrophoresis analysis demonstrated the binding of CTGF to a cell surface protein with a mass of ϳ620 kDa. We report here the purification of this protein by affinity chromatography on CTGF coupled to Sepharose and sequence information obtained by mass spectroscopy. The binding protein was identified as the multiligand receptor, low density lipoprotein receptor-related protein/␣ 2 -macroglobulin receptor (LRP). The identification of LRP as a receptor for CTGF was validated by several studies: 1) binding competition with many ligands that bind to LRP, including receptor-associated protein; 2) immunoprecipitation of CTGF-receptor complex with LRP antibodies; and 3) cells that are genetically deficient for LRP were unable to bind CTGF. Last, CTGF is rapidly internalized and degraded and this process is LRP-dependent. In summary, our data indicate that LRP is a receptor for CTGF, and may play an important role in mediating CTGF biology.

SUMMARY:Connective tissue growth factor (CTGF) is a 38-kd protein involved in several human fibrotic disorders including atherosclerosis and skin and renal fibrosis. Although it has been shown that human and experimental liver fibrosis is associated with CTGF expression through up-regulation of CTGF mRNA by hepatic stellate cells (HSC), the role of CTGF in the liver has not yet been determined. The aim of the present study was to assess the effects of CTGF on rat primary HSC and its regulation in a well-established model of in vitro liver fibrogenesis. Incubation of primary HSC with recombinant CTGF induced a significant migratory (2.3-fold, 50 ng/ml CTGF) and proliferative effect (1.8-fold, 100 ng/ml CTGF). Type I collagen mRNA expression, as assessed by a real-time RT-PCR procedure, was also increased when cells were incubated in the presence of CTGF (2-fold, 50 ng/ml). Transforming growth factor-␤1 (TGF-␤1) strongly stimulated CTGF mRNA expression, a direct mechanism observed in the absence of any intermediate protein synthesis. Furthermore, spontaneous activation of HSC plated on plastic and stimulation by vascular endothelial growth factor, lipid peroxidation products (HNE, MDA), acetaldehyde, and platelet-derived growth factor (PDGF)-BB significantly up-regulated CTGF mRNA expression in HSC. PDGF-induced CTGF stimulation might be related in part to TGF-␤1 secretion because CTGF mRNA up-regulation observed after PDGF-BB stimulation was abrogated in the presence of neutralizing TGF-␤1 antibody. In conclusion, this study extends the role of CTGF in HSC activation and suggests that CTGF up-regulation might be a central pathway during HSC activation. (Lab Invest 2002, 82:767-773).

The biological activity of transforming growth factor beta1 (TGF-beta) is controlled by its secretion as a latent complex in which it is noncovalently associated with latency-associated peptide (LAP). Activation is the extracellular process in which TGF-beta is released from LAP, and is considered to be a primary regulatory control. We recently reported rapid and persistent changes in TGF-beta immunoreactivity in conjunction with extracellular matrix remodeling in gamma-irradiated mouse mammary gland. Our hypothesis is that these specific changes in immunoreactivity are indicative of latent TGF-beta activation. In the present study, we determined the radiation dose response and tested whether a functional relationship exists between radiation-induced TGF-beta and collagen type III remodeling. After radiation exposures as low as 0.1 Gy, we detected increased TGF-beta immunoreactivity in the mammary epithelium concomitant with decreased LAP immunostaining, which are events consistent with activation. Quantitative image analysis demonstrated a significant (P=0.0005) response at 0.1 Gy without an apparent threshold and a linear dose response to 5 Gy. However, in the adipose stroma, loss of LAP demonstrated a qualitative threshold at 0.5 Gy. Loss of LAP paralleled induction of collagen III immunoreactivity in this tissue compartment. We tested whether TGF-beta mediates collagen III expression by treating animals with TGF-beta panspecific monoclonal antibody, 1D11.16, administered i.p. shortly before irradiation. Radiation-induced collagen III staining in the adipose stroma was blocked in an antibody dose-dependent manner, which persisted through 7 days postirradiation. RNase protection assay revealed that radiation-induced elevation of total gland collagen III mRNA was also blocked by neutralizing antibody treatment. These data provide functional confirmation of the hypothesis that radiation exposure leads to latent TGF-beta activation, support our interpretation of the reciprocal shift in immunoreactivity as evidence of activation, and implicate TGF-beta as a mediator of tissue response to ionizing radiation. The sensitivity of activation to low radiation doses points to a potential role for TGF-beta in orchestrating tissue response to oxidative stress. As such, radiation may be useful as a probe to delineate the consequences of latent TGF-beta activation in situ.

A growing body of data suggests that the bone marrow stroma contains a population of pluripotent cells capable of differentiating into adipocytes, osteoblasts, and lymphohematopoietic supporting cells. In this work, the murine stromal cell lines BMS2 and +/+ 2.4 have been examined as preadipocytes and adipocytes for evidence of osteoblastic gene expression. Adipocyte differentiation has been quantitated using fluorescence activated cell sorting. Within 7-10 days of adipocyte induction by treatment with glucocorticoids, indomethacin, and methylisobutylxanthine, between 40% to 50% of the cells contain lipid vacuoles and exhibit a characteristic adipocyte morphology. Based on immunocytochemistry, both the adipocytes and preadipocytes express a number of osteoblastic markers; these include alkaline phosphatase, osteopontin, collagen (I, III), bone sialoprotein II, and fibronectin. Based on biochemical assays, the level of alkaline phosphatase expression is not significantly different between preadipocyte and adipocyte cells. However, unlike rat cell lines, dexamethasone exposure causes a dose-dependent decrease in enzyme activity. The steady-state mRNA levels of the osteoblast associated genes varies during the process of adiopogenesis. The relative level of collagen I and collagen III mRNA is lower in adipocyte-induced cells when compared to the uninduced controls. Osteocalcin mRNA is detected in preadipocytes but absent in adipocytes. These data indicate that osteoblastic gene expression is detected in cells capable of undergoing adipocyte differentiation, consistent with the hypothesis that these cell lineages are interrelated.

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