Connective tissue growth factor is secreted through the Golgi and is degraded in the endosome.

Connective tissue growth factor (CTGF) is a cysteine-rich heparin-binding polypeptide that promotes proliferation, collagen synthesis, and chemotaxis in mesanchymal cells. When coinjected subcutaneously with transforming growth factor beta (TGFbeta), CTGF promotes sustained fibrosis in rats. However, little is known about the cell biology and structure/functional relationship of CTGF. In particular, no detailed characterization of the subcellular localization of CTGF has occurred, nor have sequences been identified within this protein required for this localization. In this report, using immunofluorescence and Western blot analysis, we show that CTGF is localized to the Golgi apparatus both in dermal fibroblasts and activated hepatic stellate cells. Using these methods, no CTGF was detected in endosomal, plasma membrane, cytosolic or nuclear fractions. Addition of brefeldin A, a drug that disrupts the Golgi, blocks the secretion of CTGF. We further show that the amino-terminal 37 amino acids of CTGF are sufficient to localize a heterologous protein (red fluorescent protein, RFP) to the Golgi. Although within this region of human CTGF is a N-glycosylation site, tunicamycin, which blocks N-linked glycosylation, has no significant effect on CTGF secretion. Surprisingly, mutation of a single amino acid residue, CYS-34, to alanine prevents localization of a CTGF-RFP fusion protein to the Golgi. These results are the first proof that endogenous CTGF is localized to the Golgi apparatus. Furthermore, using exogenously added (125)I-labeled CTGF, we show that CTGF is internalized and rapidly degraded in the endosome. That is, CTGF is quantitatively secreted through the golgi and is degraded in the endosome.

The low density lipoprotein receptor-related protein/alpha2-macroglobulin receptor is a receptor for connective tissue growth factor.

Connective tissue growth factor (CTGF) expression is regulated by transforming growth factor-beta (TGF-beta) 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 approximately 620 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/alpha2-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.

TGF-beta receptor expression and binding in rat mesangial cells: modulation by glucose and cyclic mechanical strain.

BACKGROUND: Transforming growth factor-beta (TGF-beta) is a causal factor in experimental glomerulosclerosis, and it mediates the increased extracellular matrix (ECM) accumulation that occurs in cultured mesangial cells (MCs) exposed to high glucose concentrations and cyclic mechanical strain. This change is associated with increased levels of TGF-beta, but may also involve alterations in receptor expression and binding. METHODS: Rat MCs cultured in media containing either 8 or 35 mM glucose were seeded into culture plates with elastin-coated flexible bottoms. Thereafter, they were subjected to cyclic stretch or static conditions and then examined for 125I-TGF-beta1 binding and expression of TGF-beta receptors at the gene and protein levels. RESULTS: Kinetic studies showed that MCs bound TGF-beta1 in a time- and concentration-dependent manner, expressing 6800 high-affinity receptors per cell, with an apparent dissociation constant (Kd) of 15.4 pM, while cross-linking analysis identified three TGF-beta receptors (betaR) corresponding to betaRI, betaRII, and betaRIII of 54, 73, and 200 kDa, respectively. Immunocytochemical studies of betaRI and betaRII protein revealed MC expression in a homogeneous, punctate distribution, whereas Northern analysis demonstrated the presence of the corresponding mRNAs. Exposure to cyclic stretching significantly increased (10%) the overall number of TGF-beta receptors, whereas ligands associated with betaRs I, II, and III also increased (25 to 50%). The finding of increased (30 to 40%) betaRI and betaRII transcript levels and immunoreactive protein (163 and 59%, respectively) in the absence of significant changes in the apparent Kd indicated that stretch-induced binding was the result of increased receptor synthesis and expression and not due to a change in binding affinity. In a similar, but more dramatic fashion, exposure to high glucose also elevated (50%) the receptor number, as well as the amount of ligands associated with betaRs I, II, and III (100 to 250%). This same treatment also increased the levels of betaRI and betaRII mRNA (30 to 40%) and the immunoreactive protein (82 and 82%, respectively), without significantly altering the binding affinity of the receptor. A concerted or synergistic effect of both stimuli was not evidenced. CONCLUSION: These results suggest that the modulation of TGF-beta receptors may be an additional control point in mediating the glucose- and mechanical force-induced increase in ECM deposition by MCs.

Latent transforming growth factor beta1 activation in situ: quantitative and functional evidence after low-dose gamma-irradiation.

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.

Transforming growth factor beta induces anchorage-independent growth of NRK fibroblasts via a connective tissue growth factor-dependent signaling pathway.

Connective tissue growth factor (CTGF) is a M(r)38,000 cysteine-rich peptide, the synthesis and secretion of which are selectively induced by transforming growth factor beta (TGF-beta). The relationship of CTGF to TGF-beta action on fibroblastic cells is not well understood. TGF-beta has the unique ability to stimulate the growth of normal fibroblasts in soft agar, a property of transformed cells. We have investigated whether CTGF can substitute for TGF-beta or whether CTGF action is essential for TGF-beta to stimulate anchorage-independent growth (AIG) of NRK fibroblasts. Our studies demonstrate that CTGF cannot induce AIG of NRK fibroblasts. However, CTGF synthesis and action are essential for the TGF-beta-induced AIG of NRK fibroblasts. Anti-CTGF antibodies specifically block TGF-beta-induced AIG but have no effect on platelet-derived growth factor or epidermal growth factor-induced growth in monolayer cultures and do not cross-react with platelet-derived growth factor or TGF-beta. Clones of NRK fibroblasts that express an antisense CTGF gene (NRK-ASCTGF), which blocks the expression of the endogenous CTGF gene, do not respond to TGF-beta in the AIG assay. The growth and morphology of the cells (NRK-ASCTGF) in monolayer culture are unaltered from the parent NRK cell line. The addition of recombinant CTGF to the NRK-ASCTGF clones in the presence of TGF-beta restores the AIG response of the cells. These studies demonstrate that the TGF-beta stimulation of NRK fibroblast AIG is dependent on events induced via the synergistic action of CTGF-dependent and CTGF-independent signaling pathways.

A single heteromeric receptor complex is sufficient to mediate biological effects of transforming growth factor-beta ligands.

Transforming growth factor beta (TGF-beta), a multifunctional cytokine that regulates a variety of biological functions, signals through a heteromeric receptor complex of the type I and type II TGF-beta receptors. The type II receptor, a transmembrane serine-threonine kinase, was cloned based on its ability to directly bind TGF-beta. Recently, a number of candidate type I TGF-beta receptors have been isolated. Although only one of these transmembrane kinases (R4) has been shown to mediate TGF-beta-dependent gene activation, others bind TGF-beta when overexpressed in COS cells. Consequently, it has been postulated that the diversity of TGF-beta responses is generated through the association of distinct type I receptors with the type II TGF-beta receptor, thus creating receptor complexes of differential signaling capacities. In contrast to this model, we demonstrate that stable expression of only the R4 type I TGF-beta receptor in a mutant cell line lacking endogenous type I TGF-beta receptor was able to complex with the endogenous type II TGF-beta receptor and restore the effects of TGF-beta on inhibition of cell proliferation and activation of specific genes, regardless of which of the three mammalian isoforms of TGF-beta was used as the ligand. Therefore, R4 acts as a fully functional type I TGF-beta receptor, and the differential effects of TGF-beta are likely mediated by a single receptor complex consisting of R4 and the type II receptor.

Effects of transforming growth factor beta 1 on growth and apoptosis of human acute myelogenous leukemia cells.

Because limited studies examined effects of transforming growth factor (TGF) beta 1 on growth of human acute myelogenous leukemia (AML) cells, we used factor-dependent and primary AML cells to assess TGF-beta 1 effects on human AML cell growth. OCI-AML1 cells were growth inhibited by TGF-beta 1 regardless of which growth factor was used as a stimulus. In contrast, AML-193 cells were resistant to TGF-beta 1 when grown with or without growth factors. UCSD/AML1 cells were sensitive to TGF-beta 1 inhibition when grown with most cytokines but were relatively resistant to TGF-beta 1 in the presence of macrophage colony-stimulating factor (M-CSF). Although cells grown from 5 of 6 AML patients were inhibited by TGF-beta 1, cells from 1 AML patient were growth stimulated by TGF-beta 1 in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF), M-CSF, or mast cell growth factor (kit ligand). Thus, 3 growth patterns with TGF-beta 1 were observed: (a) sensitivity to growth inhibition; (b) resistance; and (c) factor-dependent resistance. Further studies showed that AML-193 and UCSD/AML1 cells expressed type II TGF-beta 1 receptors and that ability of TGF-beta 1 to decrease GM-CSF receptors did not correlate with growth inhibition. AML-193 cells and UCSD/AML1 cells grown with M-CSF could be propagated in 1 ng/ml TGF-beta 1, but UCSD/AML1 cells grown with GM-CSF and TGF-beta 1 died. Morphology and agarose gel analysis of DNA showed UCSD/AML1 cells underwent apoptosis when grown with GM-CSF and TGF-beta 1 but not with M-CSF and TGF-beta 1. Similar studies of OCI-AML1 cells showed that TGF-beta 1 induced apoptosis of cells grown in 5637 bladder cell-conditioned medium or GM-CSF. These studies indicate that human AML cells exhibit heterogeneous growth responses to TGF-beta 1 and that some effects of TGF-beta 1 on myeloid cells occur through programmed cell death.

Osteoblastic gene expression during adipogenesis in hematopoietic supporting murine bone marrow stromal cells.

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.

Role of receptor complexes in resistance or sensitivity to growth inhibition by TGF beta in intestinal epithelial cell clones.

Untransformed rat intestinal epithelial cells (IEC-18) were chemically mutagenized, selected in the presence of TGF beta 1, and cloned by limiting dilution. Two clones (4-5, 4-6) were resistant to growth inhibition by both TGF beta 1 and TGF beta 2. Another clone (4-1) was more sensitive to both TGF beta isoforms (relative to parental IEC-18 cells). IC50 values for TGF beta 1 and 2 in the 4-1 cells were at least 1/9 those of the parental cells; growth rates were reduced by 49% for TGF beta 1 and by 26% for TGF beta 2 in this clone. This increased sensitivity to TGF beta was explained by the 5- to 10-fold increase, relative to parental cells, in binding of TGF beta 1 and TGF beta 2 to both the type I and II receptors. In contrast, the resistance to growth inhibition by TGF beta in the 4-5 and 4-6 cells could not be explained by a decrease in either TGF beta binding affinities or in total number of receptors expressed, by the presence of serum binding components, or by occupation of receptor binding sites with autocrine TGF-beta 1. However, in comparison to TGF beta-sensitive cells (IEC-18, 4-1), the resistant cells displayed a higher ratio of type II relative to type I receptor binding by TGF-beta 1. Thus, a critical ratio of binding to receptor subtypes correlated with growth inhibition by TGF-beta 1. Resistance to TGF-beta 2 in the same clones did not appear to be receptor related. Thus, different mechanisms for resistance to TGF-beta 1 and TGF-beta 2 were observed within a given clone.

Molecular cloning and characterization of a novel rat activin receptor.

We report the isolation of a full-length rat cDNA for a new activin receptor. The deduced amino acid sequence of this receptor shows 67 percent overall identity with that of a previously identified mouse activin receptor. As predicted for the mouse activin receptor, the amino acid sequence of the rat receptor is consistent with a polypeptide containing an extracellular ligand binding domain, a hydrophobic transmembrane domain, and a serine/threonine kinase intracellular domain. In an expression assay, this new receptor was found to bind I125 radiolabeled activin.

Two novel patterns of transforming growth factor beta (TGF-beta) binding to cell surface proteins are dependent upon the binding of TGF-beta 1 and indicate a mechanism of positive cooperativity.

Three isoforms of the transforming growth factor beta (TGF-beta) family, TGF-beta 1, TGF-beta 2, and TGF-beta 3, bind specifically and with high affinity to several cell surface components known as type I, type II, and type III proteins. The type I and II proteins may serve as biological receptors, whereas the type III protein does not appear to be associated with TGF-beta-mediated cell responses, and its function remains unknown. Binding data on confluent monolayers of rat skeletal myoblasts of the L6 cell line reveals two novel patterns of TGF-beta 1 binding. Saturation of the type I receptor with native TGF-beta 2 induces a 7-fold increase in binding of radiolabeled TGF-beta 1 at the type II protein. No induction of type II receptor binding was observed on subconfluent cells indicating a density-dependent phenomenon. The data suggest that the type I and type II proteins may interact during ligand binding in a manner which may be indicative of a regulatory role that is activated by the phase of cell growth or differentiation. A second observation is the binding of TGF-beta to a glycoprotein of 180 kDa and referred to here as the "type VI" binding protein. This protein is not related to previously described TGF-beta binding proteins, and its distribution appears universal among cell types. The level of TGF-beta 1 binding to this protein is dependent on the presence of TGF-beta 2. It is not known whether this protein transmits biological information or whether it serves as an accessory protein of a TGF-beta receptor complex.

TGF-beta receptors.

TGF-beta binds specifically and with high affinity to all cell types with few exceptions. The binding parameters vary because the dissociation constants range from 1 pM to 60 pM. There may be as few as 200 or as many as 100,000 receptors per cell, depending on the cell type. Several cell surface proteins that specifically bind TGF-beta 1 and TGF-beta 2 have been characterized as putative receptors and have been classified on the basis of their size. Type I receptors have an Mr of approximately 50,000, type II receptors an Mr of approximately 80,000. Both type I and II receptors are glycoproteins and TGF-beta-induced responses appear to be mediated by one or both types. The most abundant and largest of the cell surface TGF-beta binding proteins is a membrane-bound proteoglycan that is a dimer of subunits each with Mr of approximately 250,000 (beta-glycan). This protein is plentiful on primary cells of mesenchymal origin but absent on primary epithelial and endothelial cells. It does not appear to be associated with TGF-beta-mediated cell responses and its function remains unknown. There are multiple cellular responses to TGF-beta and accordingly there appear to be multiple pathways for signal transduction. A guanine nucleotide binding protein-dependent pathway is involved in transmission of the signal for at least one TGF-beta-induced response while there is evidence that other responses are mediated through an independent pathway. TGF-beta receptor purification and cloning efforts will be rewarded with valuable information on the mechanisms of signal transmission.

Molecular cloning of a novel bone-forming compound: osteoinductive factor.

cDNA clones encoding osteoinductive factor (OIF) have been isolated from a bovine osteoblast library. Sequence analysis of these clones indicated that the 105-amino-acid OIF is synthesized as a larger 299-amino-acid precursor, the carboxyl terminus of which is cleaved to yield the mature protein. Northern blot analysis of bovine osteoblast mRNA revealed two OIF-specific transcripts of 1.9 and 2.4 kb. The polymerase chain reaction was used to obtain clones coding for human OIF from the osteosarcoma cell line, MG-63. The human OIF cDNA encodes a precursor of 298 amino acids that exhibits 94% identity to the bovine protein. Northern blot analysis of various cell lines and tissues indicated that expression of OIF transcripts is limited and may be restricted to cells of bone lineage.

Purification and characterization of a unique osteoinductive factor from bovine bone.

A unique protein that promotes ectopic osteoinduction in the rat has been isolated and characterized. Osteoinductive factor (OIF) was extracted from the organic matrix of bovine bone with 4 M guanidine HCl and purified by gel filtration, ion-exchange chromatography, affinity chromatography, and reversed phase high performance liquid chromatography. OIF is a glycoprotein with an apparent molecular mass of 22-28 kDa based on sodium dodecyl sulfate gel electrophoresis. Enzymatic or chemical deglycosylation of OIF reduces its mass to about 12 kDa with apparent loss of activity. OIF activity in the model used is substantially increased by addition of transforming growth factor (TGF)-beta 1 or TGF-beta 2, suggesting an important role for TGF-beta 1 and -2 in bone regeneration and repair. The N-terminal sequence of OIF has no homology to other reported proteins.

Transforming growth factor beta 1 (TGF-beta 1) receptor expression on resting and mitogen-activated T cells.

Transforming growth factor beta 1 (TGF-beta 1) is a potent autocrine growth inhibitor of lymphocytes. In this study, the expression of TGF-beta 1 binding proteins was characterized on murine splenic T cells. With an affinity cross-linking method and by neutralizing antibodies to TGF-beta 1, [125I] TGF-beta 1 was found to bind to three cell surface-binding proteins (280-200 kD, 95-85 kD, 65 kD) that were differentially expressed on resting and mitogen-stimulated T cells. Freshly prepared (resting) T cells were found to constitutively express the 95-85-kD form of these binding proteins, whereas mitogenic stimulation by either concanavalin-A (Con-A), interleukin-1 (IL-1), interleukin-2 (IL-2), or 12-tetradecanoyl-phorbol-13-acetate (TPA) for 12-72 h induced the appearance of all forms of the TGF-beta 1 binding proteins (280-200 kD, 95-85 kD, and 65 kD). Furthermore, antibodies that neutralized the biologic action of TGF-beta 1 also blocked the binding of [125I] TGF-beta 1 to all three binding proteins, suggesting that these binding proteins are involved with signal transduction. These results suggest that the expression of the TGF-beta 1 receptor on T cells is regulated by T cell mitogenic signals and that a regulatory relationship may exist between T cell growth-promoting cytokines (IL-1 and IL-2) and the T cell growth inhibitor, TGF-beta 1.

Binding of transforming growth factor-beta to cell surface proteins varies with cell type.

Transforming growth factor-beta (TGF beta 1 and TGF beta 2) bind to several different cell surface proteins, including a high Mr proteoglycan. We found that on primary and early passage cultures of fibroblasts, chondroblasts, and osteoblasts TGF beta 1 binds to both the high Mr proteoglycan and to lower Mr components, whereas on epithelial, endothelial, and lymphoid-derived cells TGF beta 1 only binds to the lower Mr species. With cell lines, this distinction is lost. Further analysis indicated that binding to the high Mr proteoglycan is not necessary for TGF beta 1 induced regulation of DNA, collagen and fibronectin synthesis, change in cell morphology, or reorganization of the actin cytoskeleton. We propose that the lower Mr components are the active receptors mediating these events.