Transforming growth factor ß1 acid interaction

Chick embryo skin fibroblasts release transforming growth factor ₁ that is able to modulate glycosaminoglycan synthesis and secretion. When incubated with individual classes of glycosaminoglycans, the factor's modulatory activity was altered. To determine whether direct interactions between transforming growth factor ₁ and glycosaminoglycans occur, we have assessed the activity of the growth factor after pre-incubation with single classes of glycosaminoglycans by assaying its inhibitory effect upon the proliferative response of thymocytes stimulated with interleukin-1. Untreated transforming growth factor ₁ suppressed the proliferative response of thymocytes to interleukin-1, as did transforming growth factor ₁ pre-incubated with sulphated glycosaminoglycans. By contrast, transforming growth factor ₁ lost its inhibitory capacity when preincubated with high molecular weight hyaluronic acid. Digestion of transforming growth factor ₁-hyaluronic acid complex with hyaluronidase released active transforming growth factor ₁. Trypsin degraded transforming growth factor ₁ alone, but did not degrade the transforming growth factor ₁-hyaluronic acid complex. These results suggest that hyaluronic acid interacts with transforming growth factor ₁, thus protecting the factor from tryptic degradation and may be a means of concentrating growth factor activity.     

Latent transforming growth factor β-binding protein-3 and fibulin-1C interact with the extracellular domain of the heparin-binding EGF-like growth factor precursor

Background  

The membrane-bound cell-surface precursor and soluble forms of heparin-binding epidermal growth factor-like growth factor (HB-EGF) contribute to many cellular developmental processes. The widespread occurrence of HB-EGF in cell and tissue types has led to observations of its role in such cellular and tissue events as tumor formation, cell migration, extracellular matrix formation, wound healing, and cell adherence. Several studies have reported the involvement of such extracellular matrix proteins as latent transforming growth factor β-binding protein, TGF-β, and fibulin-1 in some of these processes. To determine whether HB-EGF interacts with extracellular matrix proteins we used the extracellular domain of proHB-EGF in a yeast two-hybrid system to screen a monkey kidney cDNA library. cDNA clones containing nucleotide sequences encoding domains of two proteins were obtained and their derived amino acid sequences were evaluated.     

Comparison of transforming growth factor β and a human tumour-derived suppressor factor

Summary Serum-free supernatants from the human melanoma cell line G361 contain a factor that can potently suppress the generation of tumouricidal lymphokine-activated killer (LAK) cells in response to interleukin-2. To characterise the suppressive factor of tumour origin we performed a number of physicochemical and functional comparisons with another immunosuppressive protein, transforming growth factor (TGF). The bioactivity of tumour-derived suppressor factor (TDSF), assayed by suppression of LAK cell generation, was unaffected by a reducing agent but lost when denatured with a chaotropic agent. In contrast, TGF was inactivated by reduction but not denaturation. TDSF lost bioactivity in conditions of pH less than 4, whereas TGF showed no loss of activity. The TDSF moiety has an estimated pI of 4.3 and a molecular mass of 69–87 kDa. This differs from published values of pI 9.5, and 25 kDa molecular mass for TGF. Anti-TGF antiserum reversed the effects of TGF but did not affect the suppression of LAK cell generation caused by TDSF. These findings provide compelling evidence that the TDSF moiety is not TGF, and may be a novel immunoregulatory cytokine.     

Hyaluronan facilitates transforming growth factor-β1-dependent proliferation via CD44 and epidermal growth factor receptor interaction

Fibroblast proliferation is an early feature of progressive tissue fibrosis and is largely regulated by the cytokine transforming growth factor-β1 (TGF-β1). In the oral mucosa, fibroblasts have a unique phenotype and demonstrate healing with no fibrosis/scarring. Our previous studies show that whereas dermal fibroblasts proliferate in response to TGF-β1, oral fibroblasts have an antiproliferative response to this cytokine. Hyaluronan (HA) was directly linked to this TGF-β1-dependent response. The aim of this study was to understand the underlying mechanism through which HA regulates TGF-β-dependent responses. Using patient-matched oral and dermal fibroblasts, we show that TGF-β1-dependent proliferation is mediated through the HA receptor CD44, whereas the TGF-β1-mediated antiproliferative response is CD44-independent. Furthermore, overexpression of HAS2 (HA synthase-2) in oral cells modifies their response, and they subsequently demonstrate a proliferative, CD44-dependent response to TGF-β1. We also show that epidermal growth factor (EGF) and its receptor (EGFR) are essential for TGF-β1/HA/CD44-dependent proliferation. Increased HA levels promote EGFR and CD44 coupling, potentiating signal transduction through the MAPK/ERK pathway. Thus, in a HA-rich environment, late ERK1/2 activation results from EGFR/CD44 coupling and leads to a proliferative response to TGF-β1. In comparison, in a non-HA-rich environment, only early ERK1/2 activation occurs, and this is associated with an antiproliferative response to TGF-β1. In summary, HA facilitates TGF-β1-dependent fibroblast proliferation through promoting interaction between CD44 and EGFR, which then promotes specific MAPK/ERK activation, inducing cellular proliferation.     

Thrombospondin-4 critically controls transforming growth factor β1 induced hypertrophic scar formation

Transforming growth factor β (TGF-β) is a growth factor presenting important functions during tissue remodeling and hypertrophic scar (HS) formation. However, the underlying molecular mechanisms are largely unknown. In this study, we identified thrombospondin-4 (TSP-4) as a TGF-β1 target that essentially mediates TGF-β1-induced scar formation both in vitro and in vivo. The expression of TSP-4 was compared on both mRNA and protein levels between hypertrophic scar fibroblasts (HSFs) and normal skin fibroblast (NFs) in response to TGF-β1 treatment. Two signaling molecules, Smad3 and p38, were assessed for their importance in regulating TGF-β1-mediated TSP-4 expression. The significance of TSP-4 in controlling TGF-β1-induced proliferation, invasion, migration, and fibrosis in HSFs was analyzed by knocking down endogenous TSP-4 using small hairpin RNA (shRNA) (TSP-4 shRNA). Finally, a skin HS model was established in rats and the scar formation was compared between rats treated with vehicle (saline), TGF-β1, and TGF-β1 + TSP-4 shRNA. The TSP-4 level was significantly higher in HSFs than in NFs and TGF-β1 more potently boosted TSP-4 expression in the former than in the latter. Both Smad3 and p38 essentially mediated TGF-β1-induced TSP-4 expression. TSP-4 shRNA significantly suppressed TGF-β1-stimulated proliferation, invasion, migration, or fibrosis of HSFs in vitro and drastically improved wound healing in vivo. TGF-β1, by activating both Smad3 and p38, induces TSP-4, which in turn not only presents a positive feedback regulation on the activation of Smad3 and p38, but also essentially mediates TGF-β1-induced HS formation. Targeting TSP-4 thus may benefit HS treatment.     

Transforming growth factor β induces expression of connective tissue growth factor in hepatic progenitor cells through Smad independent signaling

Hepatic progenitor cells (HPCs) are activated in the chronic liver injury and are found to participate in the progression of liver fibrosis, while the precise role of HPCs in liver fibrosis remains largely elusive. In this study, by immunostaining of human liver sections, we confirmed that HPCs were activated in the cirrhotic liver and secreted transforming growth factor β (TGF-β) and connective tissue growth factor (CTGF), both of which were important inducers of liver fibrosis. Besides, we used HPC cell lines LE/6 and WB-F344 as in vitro models and found that TGF-β induced secretion of CTGF in HPCs. Moreover, TGF-β signaling was intracrine activated and contributed to autonomous secretion of CTGF in HPCs. Furthermore, we found that TGF-β induced expression of CTGF was not mediated by TGF-β activated Smad signaling but mediated by TGF-β activated Erk, JNK and p38 MAPK signaling. Taken together, our results provide evidence for the role of HPCs in liver fibrosis and suggest that the production of CTGF by TGF-β activated MAPK signaling in HPCs may be a therapeutic target of liver fibrosis.     

Effects of transforming growth factor β, tumor necrosis factor α and interferon γ on pancreatic islet β-cell responsiveness to transforming growth factor α

The insulin-producing pancreatic islet -cell, characterized by low proliferative potential, is normally not responsive to the polypeptide epidermal growth factor (EGF) or its homolog transforming growth factor (TGF-). Since EGF receptors in other tissues can be up-regulated by other growth factors and by cytokines, we have in this paper investigated whether such a -cell responsiveness to TGF-, or EGF, can be conferred by co-culture with interferon (IFN-), tumor necrosis factor (TNF-) or transforming growth factor (TGF-) in various combinations. To this end, fetal rat pancreatic islets enriched in -cells were isolated and cultured for 3 days with or without 200 pM or 20 nM TGF-. It was found that neither of these TGF- concentrations affected -cell mitogenesis, insulin content or insulin secretion. However, IFN- (1000 U/ml) evoked a modest stimulation of -cell replication, while suppressing insulin secretion and leaving the islet insulin content unaltered. TNF- (1000 U/ml), on the other hand, affected none of these parameters either alone or in any combination with TGF- or IFN-. However, when TNF- or IFN-, either alone or in combination, were combined with the cytokine interleukin-1, this resulted in islet disintegration, whereas the latter cytokine alone did not exert any gross necrotic changes evident by light microscopy. TGF- (500 pM) stimulated insulin secretion but did not influence islet insulin content or -cell mitogenesis either alone or in combination with TGF- (200 pM or 20 nM). In no instance could any mitogenic or secretory response to low or high concentrations of TGF- be conferred by IFN-, TNF- or TGF- whether used alone or in combinations. Hence, responsiveness to TGF- or EGF in the -cell obviously cannot be achieved by any of these peptides.Abbreviations EGF epidermal growth factor - IFN- interferon - TGF- transforming growth factor - TGF- transforming growth factor - TNF- tumor necrosis factor      

Insulin-like growth factor 1 stimulation of androgen receptor activity requires β(1A) integrins

Despite the findings that β1 integrins play a vital role in the regulation of cell proliferation and survival, the mechanisms through which they operate and lead to cancer progression remain elusive. Previously, our laboratory has shown that β(1A) integrins support insulin-like growth factor 1 (IGFI)-mediated mitogenic and transforming activities. Here, we report that β(1A) integrins regulate basal levels of IGF-IR, although they are not critical for maintaining cancer cell morphology. Upon transfection of β(1A) siRNA and consequent downregulation of IGF-IR, we show inhibition of anchorage-independent growth of prostate cancer cells, a function which is dependent on IGF-IR expression. In addition, we demonstrate that IGFI-mediated activation of androgen receptor (AR), known to occur in prostate cancer cells, requires expression of β(1A) integrins as evaluated by luciferase reporter assays and immunoblotting analysis. Since β(1A) integrin levels are increased by R1881 or dihydrotestosterone (DHT), our results imply that β(1A) integrins support an androgen-enhanced feedback loop that regulates the expression of IGF-IR. β(1A) integrins also regulate inducible levels of IGF-IR in cells stimulated by androgen or by a combination of androgen and IGFI, as evaluated by flow cytometric analysis and immunoblotting. Furthermore, upon transfection of β(1A) siRNA and consequent downregulation of IGF-IR, neither activation of AKT, an effector of IGF-IR, nor AR levels are affected. We conclude that β(1A) integrin expression is critical for maintaining the regulatory crosstalk between IGF-IR and AR.     

Effects of transforming growth factor β1 and basic fibroblast growth factor on lipoprotein lipase activity in primary cultures of chicken (Gallus domesticus) adipocyte precursors

• 1.1. The effect of TGF-β and bFGF on lipoprotein lipase activity in chicken adipocyte precursors was investigated.
• 2.2. Lipoprotein lipase activity was reduced by up to 80% by incubation with TGF-β whereas bFGF had no effect.
• 3.3. Contrary to that found with the 3T3-L1 preadipocyte cell line it was not necessary for TGF-β to be present prior to the start of differentiation in order to be effective.
• 4.4. Incubation of adipocyte precursors with actinomycin D abolished the effect of TGF-β suggesting that synthesis of a protein effector is required.
• 5.5. These results indicate differences in responsiveness to TGF-β and bFGF between primary chicken adipocyte precursors and some preadipocyte cell lines.

     

Connective tissue growth factor induction by lysophosphatidic acid requires transactivation of transforming growth factor type β receptors and the JNK pathway

Transforming growth factor β (TGF-β) is a very strong pro-fibrotic factor which mediates its action, at least in part, through the expression of connective tissue growth factor (CTGF/CCN2). Along with these cytokines, the involvement of phospholipids in wound healing and the development of fibrosis has been revealed. Among them, lysophosphatidic acid (LPA) is a novel, potent regulator of wound healing and fibrosis that has diverse effects on many types of cells. We decided to evaluate the effect of LPA together with TGF-β on CTGF expression. We found that myoblasts treated with LPA and TGF-β1 produced an additive effect on CTGF expression. In the absence of TGF-β, the induction of CTGF expression by LPA was abolished by a dominant negative form of the TGF-β receptor type II (TGF-βRII) and by the use of SB 431542, a specific inhibitor of the serine/threonine kinase activity of TGF-βRI, suggesting that CTGF induction is dependent on LPA and requires active TGF-βRs. Moreover, we show that LPA requires Smad-2/3 proteins for the induction of CTGF expression, but not their phosphorylation or their nuclear translocation. The requirement of TGF-βRI for LPA mediated-effects is differential, since treatment of myoblasts with LPA in the presence of SB 431542 abolished the induction of stress fibers but not the induction of proliferation. Finally, we demonstrated that CTGF induction in response to LPA requires the activation of JNK, but not ERK, signaling pathways. The JNK requirement is independent of TGF-βRI-mediated activity. These novel results for the mechanism of action of LPA and TGF-β are important for understanding the role of pro-fibrotic growth factors and phospholipids involved in wound healing and related diseases.