Effects of transforming growth factor-beta 1 and fibronectin on SPARC expression in cultures of human periodontal ligament cells

Transforming growth factor-beta(1) (TGF-beta(1)) increases synthesis of secreted protein, acidic and rich in cysteine (SPARC), as well as fibronectin (FN) and type I collagen. However, little is known about the regulatory mechanism of SPARC expression. We examined the effect of FN on SPARC expression by TGF-beta(1) in cultures of human periodontal ligament cells (HPL cells). TGF-beta(1) increased the SPARC and SPARC mRNA levels in HPL cells. Extracellular matrix (ECM) produced by HPL cells in the presence of TGF-beta(1) also increased the SPARC levels. Contents of FN and type I collagen in the ECM were increased by TGF-beta(1). HPL cells cultured on FN-coated plates secreted more SPARC than those on non-coated plates. However, type I collagen had little effect on SPARC levels. The addition of anti-alpha5 antibody to the cultures abolished the increase in SPARC mRNA expression by TGF-beta(1). This study demonstrated that FN may be partly involved in the increase in SPARC expression by TGF-beta(1) in HPL cells.     

Regulation of gene expression by SPARC during angiogenesis in vitro. Changes in fibronectin, thrombospondin-1, and plasminogen activator inhibitor-1.

Angiogenesis in vitro, the formation of capillary-like structures by cultured endothelial cells, is associated with changes in the expression of several extracellular matrix proteins. The expression of SPARC, a secreted collagen-binding glycoprotein, has been shown to increase significantly during this process. We now show that addition of purified SPARC protein, or an N-terminal synthetic peptide (SPARC4-23), to strains of bovine aortic endothelial cells undergoing angiogenesis in vitro resulted in a dose-dependent decrease in the synthesis of fibronectin and thrombospondin-1 and an increase in the synthesis of type 1-plasminogen activator inhibitor. SPARC decreased fibronectin mRNA by 75% over 48 h, an effect that was inhibited by anti-SPARC immunoglobulins. Levels of thrombospondin-1 mRNA were diminished by 80%. Over a similar time course, both mRNA and protein levels of type 1-plasminogen activator inhibitor (PAI-1) were enhanced by SPARC and the SPARC4-23 peptide. The effects were dose-dependent with concentrations of SPARC between 1 and 30 micrograms/ml. In contrast, no changes were observed in the levels of either type I collagen mRNA or secreted gelatinases. Half-maximal induction of PAI-1 mRNA or inhibition of fibronectin and thrombospondin mRNAs occurred with 2-5 micrograms/ml SPARC and approximately 0.05 mM SPARC4-23. Strains of endothelial cells that did not form cords and tubes in vitro had reduced or undetectable responses to SPARC under identical conditions. These results demonstrate that SPARC modulates the synthesis of a subset of secreted proteins and identify an N-terminal acidic sequence as a region of the protein that provides an active site. SPARC might therefore function, in part, to achieve an optimal ratio among different components of the extracellular matrix. This activity would be consistent with known effects of SPARC on cellular morphology and proliferation that might contribute to the regulation of angiogenesis in vivo.     

Regulation of fibronectin and type I collagen mRNA levels by transforming growth factor-beta

Human platelet-derived transforming growth factor-beta (TGF-beta 1) increases the accumulation of the extracellular matrix proteins, fibronectin and type I collagen, in mesenchymal and epithelial cells. To determine the basis for this effect, we have examined the levels of mRNAs corresponding to fibronectin and alpha 2(I) procollagen in NRK-49 rat fibroblasts and L6E9 rat myoblasts treated with TGF-beta 1. TGF-beta 1 increased severalfold the levels of mRNAs for both proteins. The kinetics of this effect were similar for both mRNA species. The increase in fibronectin and alpha 2(I) procollagen mRNAs was detectable 2 h after addition of TGF-beta 1 to the cells and their maximal levels remained constant for several days. Actinomycin D, but not cycloheximide, inhibited the increase in fibronectin and alpha 2(I) procollagen mRNA levels induced by TGF-beta 1. The results indicate that TGF-beta 1 controls the composition and abundance of extracellular matrices at least in part by inducing a coordinate increase in the levels of fibronectin and type I collagen mRNAs.     

Matrix protein synthesis by glomerular mesangial cells in culture: Effects of transforming growth factor β (TGFβ) and platelet-derived growth factor (PDGF) on fibronectin and collagen type IV mRNA

The pathogenesis of glomerular scarring is multifactional; recent evidence suggests that transforming growth factor β (TGFβ), a pleiotropic cicatricial mediator, may promote mesangial sclerosis by enhancing the production of extracellular matrix proteins. We studied the effect of TGFβ1 and TFGβ2 on collagen type IV and fibronectin (FN) synthesis in human glomerular mesangial cells in culture (GMC). Two hours after addition of TGFβ, an up to twofold increase in abundance of collagen type IV mRNA was found, which further increased up to fivefold within 24 h. Addition of cycloheximide did not inhibit the TGFβ effect, but caused by itself an up to twofold increase in the abundance of collagen type IV mRNA after 2 h. Together with collagen mRNA, the mRNA for FN and for platelet-derived growth factor (PDGF) was also enhanced. PDGF was found to enhance abundance of the collagen type IV and fibronectin mRNA in GMC. A neutralizing antibody to PDGF or a PDGF-antisense oligonucleotide partly inhibited the TGFβ-induced increase of collagen type IV mRNA, suggesting that TGFβ can affect the collagen type IV synthesis not only directly but also indirectly via the synthesis of PDGF. © 1995 Wiley-Liss, Inc.     

Differential effects of various growth factors and cytokines on the syntheses of DNA,type I collagen,laminin, fibronectin,osteonectin/secreted protein,acidic and rich in cysteine (SPARC), and alkaline phosphatase by human pulp cells in culture

The purpose of this study is to differentiate roles of several growth factors and cytokines in proliferation and differentiation of pulp cells during development and repair. In human pulp cell cultures, laminin and type I collagen levels per cell remained almost constant during the whole culture period (22 days). On the other hand, secreted protein, acidic and rich in cysteine (SPARC/osteonectin) and alkaline phosphatase (ALPase) levels markedly increased after the cultures reached confluence. Laminin and type I collagen, as well as fibronectin, stimulated the spreading of pulp cells within 1 h. Adding transforming growth factor-β (TGF-β) decreased laminin and ALPase levels, whereas it increased SPARC and fibronectin levels 3- to 10-fold. Western and Northern blots showed that TGF-β enhanced SPARC synthesis at the protein and mRNA levels. Basic fibroblast growth factor (bFGF) decreased type I collagen, laminin, SPARC, and ALPase levels without changing the fibronectin level. Platelet-derived growth factor (PDGF) selectively decreased laminin, SPARC, and ALPase levels. Epidermal growth factor (EGF) also decreased SPARC and ALPase levels. Tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) decreased type I collagen and laminin levels, and abolished SPARC and ALPase syntheses. Of these peptides, bFGF and PDGF showed the greatest stimulation of [³H]thymidine incorporation into DNA. TGF-β, EGF, and TNF-α had less effect on DNA synthesis, whereas IL-1β inhibited DNA synthesis. These findings demonstrated that TGF-β, bFGF, EGF, PDGF, TNF-α, and IL-1β have characteristically different patterns of actions on DNA, laminin, type I collagen, fibronectin, ALPase, and SPARC syntheses by pulp cells. J. Cell. Physiol. 174:194–205, 1998. © 1998 Wiley-Liss, Inc.     

Mice lacking serum amyloid P component do not necessarily develop severe autoimmune disease

Interleukin-10 (IL-10) is a cytokine with many regulatory functions. In particular, IL-10 exerts neutralizing effect on other cytokines, and therefore IL-10 is thought to have important therapeutic implications. Recent reports suggest that IL-10 regulates not only immunocytes but also collagen and collagenase gene expression in fibroblasts. In this study, we investigated the effect of IL-10 on gene expression of extracellular matrix (ECM) proteins, such as type I collagen, fibronectin, and decorin, in human skin fibroblasts. Results of Northern blot analysis showed that both collagen I and fibronectin mRNAs were downregulated, while decorin gene expression was enhanced by IL-10 (10 ng/ml) time-dependently (6-24 h). alpha1(I) collagen and fibronectin mRNAs were decreased to one-third and one-fourth, respectively, by 50 ng/ml IL-10, whereas decorin mRNA was increased up to 2.7-fold by 50 ng/ml IL-10. Response to IL-10 by scleroderma fibroblasts was similar to that in normal dermal fibroblasts, with decreased expression levels of collagen and fibronectin and induced decorin mRNA levels. Transforming growth factor-beta (TGF-beta) is a crucial fibrogenic cytokine which upregulates the mRNA expression of collagen and fibronectin, whereas it downregulates decorin mRNA expression in fibroblasts. Monocyte chemoattractant protein-1 (MCP-1) has recently been shown to upregulate the type I collagen mRNA expression in cultured fibroblasts. We therefore examined whether IL-10 alters gene expression of ECM elicited by TGF-beta and MCP-1. Our results demonstrated that IL-10 downregulated the TGF-beta-elicited increase of mRNA expression of type I collagen and fibronectin, while partially recovering TGF-beta-elicited decrease of decorin expression in normal skin fibroblasts. By contrast, IL-10 did not alter the MCP-1-elicited upregulation of mRNA expression of either alpha1(I) collagen and decorin. Our data indicate that IL-10 differentially regulates TGF-beta and MCP-1 in the modulation of ECM proteins and therefore suggest that IL-10 plays a role in the regulation of tissue remodeling.     

Transforming growth factor-beta 1 stimulates fibronectin production in bovine adrenocortical cells in culture.

Transforming growth factor-beta (TGF-beta 1) suppresses cortisol production when added to cultured bovine adrenocortical (BAC) cells while concomitantly increasing fibronectin synthesis and assembly into extracellular fibrils. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of gelatin-Sepharose-treated media from BAC cells demonstrated a 2-fold stimulation of fibronectin production by TGF-beta 1 in both the presence and absence of serum. Indirect immunofluorescence studies revealed that TGF-beta 1 caused a striking increase in the fibronectin content of BAC extracellular matrix. TGF-beta 1 caused a 4-fold increase in deoxycholate-insoluble fibronectin after 12 h and a 7-fold increase after 24 h over that of control BAC cultures. Northern hybridization analyses indicated that TGF-beta 1 stimulated levels of fibronectin poly(A)+ RNA 2.3-fold. We found that cultured BAC cells express TGF-beta 1 mRNA, suggesting a possible autocrine role for TGF-beta 1 in the adrenal.     

Transforming growth factor beta stimulates the expression of fibronectin and of both subunits of the human fibronectin receptor by cultured human lung fibroblasts

Transforming growth factors of the beta-class (TGFs-beta) stimulate extracellular matrix synthesis and have been implicated in embryogenesis, wound healing, and fibroproliferative responses to tissue injury. Because cells communicate with several extracellular matrix components via specific cell membrane receptors, we hypothesized that TGFs-beta may also regulate the expression of such receptors. We confirmed that TGF-beta 1 increases the expression of fibronectin, an adhesive glycoprotein expressed during embryogenesis and tissue remodeling. Based upon the 48-72-h period required for a maximal fibroproliferative response to dermal injections of TGF-beta 1, we exposed human fetal lung fibroblasts (IMR-90) to TGF-beta 1 for periods up to 48 h in vitro. We observed as much as 6-fold increases in fibronectin synthesis by 24 h as previously reported for fibroblastic cells (Ignotz, R. A., and Massagué, J. (1986) J. Biol. Chem. 261, 4337-4345; Ignotz, R. A., Endo, T., and Massagué, J. (1987) J. Biol. Chem. 262, 6443-6446; Raghow, R., Postlethwaithe, A. E., Keski-Oja, J., Moses, H. L., and Kang, A. H. (1987) J. Clin. Invest. 79, 1285-1288), but up to 30-fold increases by 48 h. These increases are accompanied by similar increases in fibronectin mRNA levels which are prevented by actinomycin D treatment. Using a monospecific antibody raised to the human placental fibronectin receptor complex, we found that TGF-beta 1 stimulated fibronectin receptor synthesis up to 20-40-fold and increases mRNA levels encoding both the alpha- and beta-subunits up to 3-fold, compared to control IMR-90 in serum-free medium. Actinomycin D blocks TGF-beta 1-mediated increases in receptor mRNA levels. The earliest detectable TGF-beta 1-mediated increases in fibronectin receptor complex protein synthesis and mRNA levels occur at 8 h, whereas the earliest increases in fibronectin protein synthesis and mRNA levels occur at 12 h. These results demonstrate that TGF-beta 1 stimulates fibronectin receptor synthesis, extending the diverse stimulatory activities of this polypeptide to matrix receptors. In addition, because fibronectin matrix assembly may involve the fibronectin cell adhesive receptor complex, increased receptor expression may help drive fibronectin deposition into matrix.     

Differential effects of growth factors and cytokines on the synthesis of SPARC, DNA, fibronectin and alkaline phosphatase activity in human periodontal ligament cells

Growth factors and cytokines play an important role in tissue development and repair. However, it remains unknown how they act on proliferation and differentiation of periodontal ligament cells. In this study, we investigated the effects of several growth factors and cytokines on the synthesis of DNA, alkaline phosphatase (ALPase), fibronectin, and secreted protein acidic and rich in cysteine (SPARC) in human periodontal ligament (HPL) cells. Transforming growth factor-beta (TGF-beta) increased the synthesis of DNA, fibronectin and SPARC, whereas it decreased ALPase activity. Basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF) and tumor necrosis factor-alpha (TNF-alpha) decreased SPARC and ALPase levels, whereas these peptides increased DNA synthesis and did not affect fibronectin synthesis. Epidermal growth factor (EGF) up-regulated the synthesis of DNA and fibronectin and inhibited SPARC and ALPase levels. Interleukin-1beta (IL-1beta) decreased the synthesis of DNA, ALPase, fibronectin and SPARC. These findings demonstrate that TGF-beta, bFGF, EGF, PDGF, TNF-alpha and IL-1beta have characteristically different patterns of action on DNA, SPARC, fibronectin and ALPase synthesis by HPL cells. The differences in regulation of function of periodontal ligament cells by these peptides may be involved in the regeneration and repair of periodontal tissue.     

Differential effects of transforming growth factor-beta on the synthesis of extracellular matrix proteins by normal fetal rat calvarial bone cell populations

To determine the effects of transforming growth factor-beta (TGF-beta) on the different cell types that exist in bone, cell populations (I-IV), progressively enriched in osteoblastic cells relative to fibroblastic cells, were prepared from fetal rat calvaria using timed collagenase digestions. TGF-beta did not induce anchorage-independent growth of these cells, nor was anchorage-dependent growth stimulated in most populations studied, despite a two- to threefold increase in the synthesis of cellular proteins. In all populations the synthesis of secreted proteins increased 2-3.5-fold. In particular, collagen, fibronectin, and plasminogen activator inhibitor synthesis was stimulated. However, different degrees of stimulation of individual proteins were observed both within and between cell populations. A marked preferential stimulation of plasminogen activator inhibitor was observed in each population, together with a slight preferential stimulation of collagen; the effect on collagen expression being directed primarily at type I collagen. In contrast, the synthesis of SPARC (secreted protein acidic rich in cysteine/osteonectin was stimulated approximately two-fold by TGF-beta, but only in fibroblastic populations. Collectively, these results demonstrate that TGF-beta stimulates matrix production by bone cells and, through differential effects on individual matrix components, may also influence the nature of the matrix formed by different bone cell populations. In the presence of TGF-beta, osteoblastic cells lost their polygonal morphology and alkaline phosphatase activity was decreased, reflecting a suppression of osteoblastic features. The differential effects of TGF-beta on bone cell populations are likely to be important in bone remodeling and fracture repair.     

Type X collagen gene expression is transiently up-regulated by retinoic acid treatment in chick chondrocyte cultures

During endochondral ossification, resting and proliferating chondrocytes mature into hypertrophic chondrocytes that initiate synthesis of type X collagen. The mechanisms regulating the differential expression of type X collagen gene were examined in confluent Day 12 secondary cultures of chick vertebral chondrocytes in monolayer treated with the vitamin A analog retinoic acid (RA). Preliminary results showed that major effects of RA on chondrocyte gene expression occurred between 24 and 48 h of treatment. Thus in subsequent experiments cultures were treated for 24, 30, 36, 42, 48, 72, 96, and 120 h. Total RNAs were isolated and analyzed by hybridization with ³²P-labeled plasmid probes coding for five matrix macromolecules including type X collagen. We found that the steady-state levels of mRNAs for the large keratan sulfate/chondroitin sulfate proteoglycan (KS:CS-PG) core protein and type II collagen decreased several fold between 24 and 48 h of treatment compared to untreated cells, and remained low with further treatment. In sharp contrast, the level of type X collagen mRNA increased threefold by 42 h of treatment; thereafter it began to decrease and reached minimal levels by 72–120 h of treatment. The changes in steady-state mRNA levels during RA regimen paralleled similar changes in relative rates of protein synthesis. The transient up-regulation of type X collagen gene expression at 42 h of treatment was preceded by a five-fold increase in fibronectin gene expression, was followed by a several fold increase in type I collagen gene expression, and was accompanied by cell flattening and loss of the pericellular proteoglycan matrix. Thus, RA treatment leads to a unique biphasic modulation of type X collagen gene expression in maturing chondrocyte cultures. The underlying, RA-sensitive mechanisms effecting this modulation may reflect those normally regulating the differential expression of this collagen gene during endochondral ossification.