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P R Segarini

Publications and source records attributed to P R Segarini.

18 recordsLinked to original sources
PubMed2001

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.

Amino Acid Sequence↗
PubMed1999

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.

Activin Receptors, Type I↗
PubMed1997

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.

Animals↗
PubMed1994

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.

Animals↗
PubMed1993

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.

Animals↗
PubMed1993

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.

Adipose Tissue↗
PubMed1992

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.

Affinity Labels↗
PubMed1991

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.

Animals↗
PubMed1989

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.

Amino Acid Sequence↗
PubMed1989

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.

Animals↗
PubMed1988

The high molecular weight receptor to transforming growth factor-beta contains glycosaminoglycan chains.

Proteoglycans are constituents of the cell surface that may play important roles in the regulation of cell behavior. Here we report that the 250-kDa receptor subunit that binds the multifunctional protein, transforming growth factor-beta 1 (TGF-beta 1), contains chains of heparan sulfate and chondroitin sulfate and thus is a proteoglycan. Digestion of TGF-beta 1-receptor complexes with glycosaminoglycan (GAG)-specific degradative enzymes yield core proteins of 115-140 kDa. Cell monolayers that had been predigested with GAG-specific degradative enzymes were capable of binding high levels of TGF-beta 1, but the size of the binding components was shifted from the high molecular weight species to the lower molecular weight core proteins, indicating that GAG chains are not necessary for TGF-beta 1 binding to the cell. The presence of GAG chains on the receptor subunit indicates that it has the potential for interaction with the extracellular matrix.

Animals↗
PubMed1987

Membrane binding characteristics of two forms of transforming growth factor-beta.

Cartilage-inducing factors A and B (CIF-A and CIF-B) from bovine bone have recently been identified as transforming growth factor-beta (TGF-beta) (Seyedin, S.M., Thompson, A. Y., Bentz, H., Rosen, D. M., McPherson, J. M., Conti, A., Siegel, N. R., Galluppi, G. R., and Piez, K. A. (1986) J. Biol. Chem., 261, 5693-5695) and a unique protein homologous to TGF-beta (Seyedin S. M., Segarini, P. R., Rosen, D. M., Thompson, A. Y., Bentz, H., and Graycar, J. (1987) J. Biol. Chem., 262, 1946-1949), respectively. Although the biological activities of TGF-beta and CIF-B are similar, the divergence of CIF-B from the highly conserved amino acid sequence of TGF-beta prompted an investigation of its receptor binding properties. Three classes of cell surface binding components were identified. Class A has exclusive affinity for TGF-beta; class B has greater affinity for CIF-B; and class C has equal affinity for both proteins. A high molecular weight component, the predominant binding species, was further characterized and shown to consist of two components that are either class B or class C. The differential binding properties of TGF-beta and CIF-B to cell surface components suggest that there are biological activities unique to each of the proteins.

Binding, Competitive↗
PubMed1987

Cartilage-inducing factor-B is a unique protein structurally and functionally related to transforming growth factor-beta.

Cartilage-inducing factors-A (CIF-A) and -B (CIF-B), purified from bovine bone on the basis of their ability to induce the cartilage phenotype in vitro, are proteins with molecular weights of 26,000 composed of two apparently identical disulfide-linked chains. CIF-A is apparently identical to TGF-beta from human platelets (Seyedin S. M., Thompson, A. Y., Bentz, H., Rosen, D. M., McPherson, J. M., Conti, A., Siegel, N. R., Galluppi, G. R., and Piez, K. A. (1986) J. Biol. Chem. 261, 5693-5695). We have now found that, like CIF-A and TGF-beta, CIF-B induces anchorage-independent proliferation of NRK-49F cells when these cells are simultaneously treated with epidermal growth factor. Furthermore, CIF-B competes with CIF-A for the same cell membrane receptors in NRK-49F cells. Partial amino acid sequencing reveals that CIF-B is a distinct molecule with extensive homology to CIF-A/TGF-beta. These results show that CIF-B and TGF-beta are structurally and functionally similar molecules, but differ more from each other than does TGF-beta from different species.

Amino Acid Sequence↗
PubMed1983

The centriolar antigen expression in TC7 cells is dependent on growth conditions and occurs at a particular time point in G1.

The correlation between growth conditions and centriolar antigen (Cag) expression in TC7 cells, a subline of African green monkey kidney cells, was studied. TC7 cells became quiescent when their number reached a high cell density, or when serum factors were depleted from media containing a low concentration of fetal bovine serum (FBS). There was a stoichiometric relationship between the concentration of serum present and the number of new cells produced. During proliferation, the projected cell area decreased as a function of cell density with two abrupt transitions. The first transition appeared to be independent of cell-cell contact. However, the second transition seemed to occur mainly as a result of the limitation of the available substratum surface on which the cells could grow. The appearance of Cag in TC7 cells was found to be associated with the cells' growth conditions as well as with the particular phase in cell cycle. In an exponentially growing culture of cells with 10% FBS and in cells that were growth restricted due to a high density (above 2-3 X 10(5) cells/cm2), the incidence of cells with Cag-positive staining was about 10-20%. It increased, however, to about 40-60% at cell densities between 2 X 10(4) cells/cm2 and 1 X 10(5) cells/cm2. The frequency of cells with positive Cag staining was as high as 80% in TC7 cells that were growth restricted by depletion of serum factor(s). Thus, the quiescent states attained by the two different growth restrictions seem to be different in their ability to express Cag. The frequency of Cag could be further increased by stimulating the quiescent cell population by FBS. In mitotically selected TC7 cells, Cag staining appeared about 4 hr after mitosis and about 2-4 hr before the onset of DNA synthesis. Thus, expression of Cag in TC7 cells is related to their growth conditions, and is characteristic of a part of the G1 phase of the cell cycle.

Animals↗