Induction of chondrogenesis and expression of superficial zone protein (SZP)/lubricin by mesenchymal progenitors in the infrapatellar fat pad of the knee joint treated with TGF-beta1 and BMP-7

Superficial zone protein (SZP) is a key mediator of boundary lubrication of articular cartilage in joints. In this investigation, we made the unexpected discovery that SZP was expressed in infrapatellar fat pad (IFP) from bovine knee. Quantitative analysis of secreted proteins in the medium of the IFP stromal cells demonstrated a significant stimulation by TGF-β1 and BMP-7. Real-time PCR analysis revealed the SZP expression was up-regulated by TGF-β1 and BMP-7. Chondrogenically differentiated IFP progenitor cells were stimulated by TGF-β1 and BMP-7 to synthesize and secrete SZP. SZP mRNA was significantly up-regulated by chondrogenic induction for 21 days. These findings indicate that the stimulation of SZP expression by TGF-β and BMP-7 may lead to functional improvement of damaged intraarticular tissues and that IFP progenitor cells may be a potential useful source for inducing superficial zone of articular cartilage by tissue engineering for regeneration of damaged articular cartilage due to osteoarthritis     

IL-13 induces connective tissue growth factor in rat hepatic stellate cells via TGF-β-independent Smad signaling

Connective tissue growth factor (CTGF) plays a central role in stimulating extracellular matrix deposition in the liver, and hence is considered a critical mediator of TGF-β-dependent fibrogenesis. Hepatic stellate cells (HSCs) are known as the major source of CTGF in damaged liver. However, previous studies revealed that IL-13, rather than TGF-β, represents the predominant inducer of CTGF expression in HSCs. We now dissected IL-13 downstream signaling that modulates CTGF expression in HSCs. IL-13 induces a time- and dosage-dependent increase of CTGF in a TGF-β-independent manner. This process requires participation of different Smad proteins and their upstream receptor kinases (activin receptor-like kinases). Smad1 and Smad2 were identified as the key mediators of IL-13-dependent CTGF expression. Furthermore, IL-13 induces Stat6 phosphorylation in HSCs, but Stat6 was not involved in CTGF induction. Instead, the Erk1/2-MAPK pathway was found to be responsible for IL-13-induced early Smad phosphorylation and CTGF synthesis. We demonstrate that IL-13 induces CTGF expression in HSCs by activating TGF-β-independent activin receptor-like kinase/Smad signaling via the Erk-MAPK pathway rather than via its canonical JAK/Stat6 pathway. These results provide an improved new insight into the molecular mechanisms of profibrotic IL-13 activities in the liver.     

TGF-β1-induced collagen promotes chicken ovarian follicle development via an intercellular cooperative pattern

Follicle development is a complex process under strict regulation of diverse hormones and cytokines including transforming growth factor β (TGF-β) superfamily members. TGF-β is pivotal for the regulation of ovarian functions under physiological and pathological conditions. In this study, effect of TGF-β1 on chicken follicle development was examined through investigating the accumulation and action of collagen, an indispensable member of the extracellular matrix (ECM) involved in this process. The granulosa cells (GCs) and theca cells (TCs) were separated from growing follicles of the laying chicken for treatment of TGF-β1 and analysis of expression of ECM components and key proteins in intracellular signaling pathways. Results showed that collagen was mainly distributed in the follicular theca layer and was produced with the formation of the granulosa layer during ovarian development. Collagen accumulation increased with follicle growth and treatment of GCs with TGF-β1 elicited an increased expression of collagen. After production from GCs, collagen was transferred to the neighboring TCs to promote cell proliferation and inhibit apoptosis. Treatment of collagen remarkably increased expression of p-ERK, mitogen-activated protein kinase (MAPK), and p-MAPK, but treatment with hydroxylase inhibitor (to break collagen structure) reversed these alterations. In conclusion, during follicle growth collagen was secreted by GCs under TGF-β1 stimulation and was subsequently collaboratively transferred to neighboring TCs to increase cell proliferation and thus to promote follicle development via an intercellular cooperative pattern during development of chicken growing follicles.     

Changes in TGF-β receptors of rat hepatocytes during primary culture and liver regeneration: Increased expression of TGF-β receptors associated with increased sensitivity to TGF-β-mediated growth inhibition

To clarify the role of transforming growth factor-β (TGF-β) and its receptors in hepatocyte growth, we studied the expression of TGF-β1 and its receptors and the sensitivity to growth inhibition by TGF-β1 protein in rat hepatocytes derived from resting and regenerating livers. In hepatocytes derived from resting livers, mRNAs for TGF-β type II receptor (TβR-II), insulin-like growth factor-II/mannose 6-phosphate receptor (IGF-II/M-6-PR), and TGF-β1 increased with time in primary culture. The cell surface TGF-β receptor proteins (TβR-I, II, and III), examined by the receptor affinity-labeling assay using ¹²⁵I-TGF-β1, also increased, especially after 48 hr of culture. Hepatocytes were more sensitive to inhibition of DNA synthesis, when the TGF-β1 protein was added at later times in culture, corresponding to the presence of increased TGF-β receptors. In hepatocytes from regenerating livers after a partial hepatectomy (PH), an increase of TβR-I, TβR-II, TβR-III, IGF-II/M-6-PR, and TGF-β1 mRNAs was found, compared with hepatocytes from resting livers. Similarly, using TGF-β receptor affinity-labeling assay, hepatocytes from PH livers were found to have an increase in TβR-I, II, and III proteins, with a peak at 4 days post-PH, compared with hepatocytes from resting livers. When TGF-β1 protein was added for a short period (6 or 24 hr) after cell attachment to hepatocyte cultures, it inhibited DNA synthesis more effectively in hepatocytes from regenerating compared with resting livers. Our results show that hepatocyte TGF-β receptors and sensitivity to growth inhibition by TGF-β1 protein change together and are modulated during liver regeneration, as well as during the conditions of primary culture. J. Cell. Physiol. 176:612–623, 1998. © 1998 Wiley-Liss, Inc.     

Adverse effects of adenovirus-mediated gene transfer of human transforming growth factor beta 1 into rabbit knees

To examine the effect of transforming growth factor (TGF)-β1 on the regulation of cartilage synthesis and other articular pathologies, we used adenovirus-mediated intra-articular gene transfer of TGF-β1 to both naïve and arthritic rabbit knee joints. Increasing doses of adenoviral vector expressing TGF-β1 were injected into normal and antigen-induced arthritis rabbit knee joints through the patellar tendon, with the same doses of an adenoviral vector expressing luciferase injected into the contralateral knees as the control. Intra-articular injection of adenoviral vector expressing TGF-β1 into the rabbit knee resulted in dose-dependent TGF-β1 expression in the synovial fluid. Intra-articular TGF-β1 expression in both naïve and arthritic rabbit knee joints resulted in significant pathological changes in the rabbit knee as well as in adjacent muscle tissue. The observed changes induced by elevated TGF-β1 included inhibition of white blood cell infiltration, stimulation of glycosaminoglycan release and nitric oxide production, and induction of fibrogenesis and muscle edema. In addition, induction of chondrogenesis within the synovial lining was observed. These results suggest that even though TGF-β1 may have anti-inflammatory properties, it is unable to stimulate repair of damaged cartilage, even stimulating cartilage degradation. Gene transfer of TGF-β1 to the synovium is thus not suitable for treating intra-articular pathologies.     

Activation of latent TGFβ by αvβ1 integrin: of potential importance in myofibroblast activation in fibrosis

Cell-mediated activation of latent TGF-β1 is intimately involved with tissue repair and fibrosis in all organs. Previously, it was shown that the integrin β1 subunit was required for activation of latent TGF-β1 and skin fibrosis. A recent study by Henderson and colleagues (Nature Medicine 19,1617–1624, 2013) used three different in vivo models of fibrosis to show that integrin αv subunit was required for fibrogenesis. Through a process of elimination, the authors conclude that in vivo, the little-studied α_vβ₁ could be the major integrin responsible for TGF-β activation by myofibroblasts. Thus targeting this integrin might be a useful therapy for fibrosis.     

Interleukin-20 targets renal cells and is associated with chronic kidney disease

Interleukin (IL)-10 is an anti-inflammatory factor that suppresses renal fibrosis and improves renal function in CKD rats. IL-20 belongs to the IL-10 family; therefore, we sought to determine whether IL-20 is involved in CKD. CKD patients at stage five expressed significantly higher IL-20 in serum than controls. Immunohistochemical staining demonstrated that more IL-20 protein was expressed in the kidney tubular-epithelial cells, mesangial cells, and immune cells of CKD rats with a 5/6 nephrectomy. The lung, liver, and heart tissue of CKD rats also overexpressed IL-20. Thus, we treated two tubular epithelial cells, TKPTS and M-1 cells, with IL-20 to study its effects on CKD. IL-20 treatment induced apoptosis in these cells via caspase-3 activation. Incubating IL-20 with rat interstitial fibroblasts, NRK-49F cells, upregulated TGF-β1production, one key inducer for renal fibrogenesis. Therefore, IL-20 injured renal epithelial cells and induced fibroblasts to produce TGF-β1 that hastened the progression of CKD.     

Regulation of TGF-β storage and activation in the human idiopathic pulmonary fibrosis lung

Idiopathic pulmonary fibrosis (IPF) is a progressive disease of unknown cause. The pathogenesis of the disease is characterized by fibroblast accumulation and excessive transforming growth factor-β (TGF-β) activation. Although TGF-β activation is a complex process involving various protein interactions, little is known of the specific routes of TGF-β storage and activation in human lung. Here, we have systematically analyzed the expression of specific proteins involved in extracellular matrix targeting and activation of TGF-β. Latent TGF-β-binding protein (LTBP)-1 was found to be significantly upregulated in IPF patient lungs. LTBP-1 expression was especially high in the fibroblastic foci, in which P-Smad2 immunoreactivity, indicative of TGF-β signaling activity, was less prominent. In cultured primary lung fibroblasts and epithelial cells, short-interfering-RNA-mediated downregulation of LTBP-1 resulted in either increased or decreased TGF-β signaling activity, respectively, suggesting that LTBP-1-mediated TGF-β activation is dependent on the cellular context in the lung. Furthermore, LTBP-1 was shown to colocalize with fibronectin, fibrillin-1 and fibrillin-2 proteins in the IPF lung. Fibrillin-2, a developmental gene expressed only in blood vessels in normal adult lung, was found specifically upregulated in IPF fibroblastic foci. The TGF-β-activating integrin β8 subunit was expressed at low levels in both control and IPF lungs. Alterations in extracellular matrix composition, such as high levels of the TGF-β storage protein LTBP-1 and the re-appearance of fibrillin-2, probably modulate TGF-β availability and activation in different pulmonary compartments in the fibrotic lung.     

Relationship between actions of transforming growth factor (TGF)-β and cell surface expression of its receptors in clonal osteoblastic cells

Various osteoblastic cell lines were examined for the relationship between the presence of cell-surface transforming growth factor (TGF)-β receptors and the synthesis of matrix proteins with their responsiveness to TGF-β. Treatment with TGF-β1 inhibited proliferation and stimulated proteoglycan and fibronectin synthesis in MC3T3-E1 and MG 63 cells. The major proteoglycans synthesized by these cells were decorin and biglycan, and TGF-β1 markedly stimulated the synthesis of decorin in MC3T3-E1 and of biglycan in MG 63 cells. SaOS 2 and UMR 106 cells synthesized barely detectable amounts of decorin or biglycan, and TGF-β1 did not stimulate the synthesis of these proteoglycans. In SaOS 2 cells, however, TGF-β1 enhanced fibronectin synthesis. TGF-β1 did not show any of these effects in UMR 106 cells. Receptor cross-linking studies revealed that only MC3T3-E1 and MG 63 cells had both types I and II signal-transducing receptors for TGF-β in addition to betaglycan. SaOS 2 cells possessed type I but no type II receptor on the cell surface. In contrast, SaOS 2 as well as MC3T3-E1 and MG 63 cells expressed type II receptor mRNA by Northern blot analysis, and cell lysates contained type II receptor by Western blot analysis. Thus, it appears that type II receptor present in SaOS 2 cells is not able to bind TGF-β1 under these conditions. UMR 106 cells with no response to TGF-β1 had neither of the signal-transducing receptors by any of the analyses. These observations using clonal osteoblastic cell lines demonstrate that the ability of osteoblastic cells to synthesize bone matrix proteoglycans is associated with the responsiveness of these cells to TGF-β1, that the responsiveness of osteoblastic cells to TGF-β1 in cell proliferation and proteoglycan synthesis correlates with the presence of both types I and II receptors, and that the effect of TGF-β1 on fibronectin synthesis can develop with little binding of TGF-β1 to type II receptor if type I receptor is present. It is suggested that the combination of cell-surface receptors for TGF-β determines the responsiveness of osteoblastic cells to TGF-β and that changes in cell-surface TGF-β receptors may play a role in the regulation of matrix protein synthesis and bone formation in osteoblasts. © 1995 Wiley-Liss, Inc.     

Oncostatin M inhibits TGF-β1-induced CTGF expression via STAT3 in human proximal tubular cells

Matricellular proteins play a critical role in the development of tubulointerstitial fibrosis and renal disease progression. Connective tissue growth factor (CTGF/CCN2), a CCN family member of matricellular proteins, represents an important mediator during development of glomerular and tubulointerstitial fibrosis in progressive kidney disease. We have recently reported that oncostatin M (OSM) is a potent inhibitor of TGF-β1-induced CTGF expression in human proximal tubular cells (PTC). In the present study we examined the role of TGF-β1- and OSM-induced signaling mechanisms in the regulation of CTGF mRNA expression in human proximal tubular HK-2 cells. Utilizing siRNA-mediated gene silencing we found that TGF-β1-induced expression of CTGF mRNA after 2h of stimulation at least partially depends on SMAD3 but not on SMAD2. In contrast to TGF-β1, OSM seems to exert a time-dependent dual effect on CTGF mRNA expression in these cells. While OSM led to a rapid and transient induction of CTGF mRNA expression between 15min and 1h of stimulation it markedly suppressed basal and TGF-β1-induced CTGF mRNA levels thereafter. Silencing of STAT1 or STAT3 attenuated basal CTGF mRNA levels indicating that both STAT isoforms may be involved in the regulation of basal CTGF mRNA expression. However, knockdown of STAT3 but not STAT1 prevented OSM-mediated suppression of basal and TGF-β1-induced upregulation of CTGF mRNA expression. Together these results suggest that the inhibitory effect of OSM on TGF-β1-induced CTGF mRNA expression is mainly driven by STAT3, thereby providing a signaling mechanism whereby OSM may contribute to tubulointerstitial protection.     

Transforming growth factor-β1b: A second TGF-β1 paralogue in the rainbow trout (Oncorhynchus mykiss) that has a lower constitutive expression but is more responsive to immune stimulation

The rainbow trout (Oncorhynchus mykiss) TGF-β1 sequence was one of the first fish cytokines described. Studies of its expression suggest it is constitutively expressed but displays refractory inducibility. Here we describe a second TGF-β1 (TGF-β1b) gene that is novel in several respects. TGF-β1b possesses typical TGF-β features, including a CXC motif and an integrin binding site, a tetrabasic cut site and a mature peptide of 112 amino acids (aa) containing nine conserved cysteine residues. The mature peptide is 83% identical to the first TGF-β1 sequence described in rainbow trout, that we designate TGF-β1a, and relative to TGF-β1a shows higher homology to Atlantic salmon TGF-β1b, zebrafish TGF-β1a, and sea bass and seabream TGF-β1. The gene organisation of salmonid TGF-β1b genes, as inferred from Atlantic salmon whole genome shotgun contigs, is a 6 exon/5 intron structure with exons 3 and 4 of salmonid TGF-β1a genes apparently fused together. The two trout TGF-β1 genes have a wide distribution in vivo, with highest expression found in immune tissues for both isoforms indicating that TGF-β1 has a predominant role in immunity of fish. Expression of both genes was also seen during the ontogeny of trout, with TGF-β1a relatively constant in expression level but TGF-β1b increasing over time. Immune responses in head kidney (HK) macrophages induced by pathogen associated molecular patterns (PAMPs), pro-inflammatory cytokines, mitogens and pathway activators highly elevated the expression level of TGF-β1b but not that of TGF-β1a. TGF-β1b expression was also increased by polyinosinic:polycytidylic acid (poly(I:C)) and/or lipopolysaccharide (LPS) stimulation in three different trout cell lines studied. Finally we show that TGF-β1b is potentially involved in defense against infection with viral haemorrhagic septicemia virus (VHSV), which had no effect on TGF-β1a expression. Thus, it is likely the TGF-β1b gene represents a copy which fulfils the major immune orchestrating functions of TGF-β1 as seen in other vertebrates.     

TGF-β2 secretion from RPE decreases with polarization and becomes apically oriented

Retinal pigmented epithelium (RPE) secretes transforming growth factor beta 1 and 2 (TGF-β1 and -β2) cytokines involved in fibrosis, immune privilege, and proliferative vitreoretinopathy (PVR). Since RPE cell polarity may be altered in various disease conditions including PVR and age-related macular degeneration, we determined levels of TGF-β from polarized human RPE (hRPE) and human stem cell derived RPE (hESC-RPE) as compared to nonpolarized cells. TGF-β2 was the predominant isoform in all cell culture conditions. Nonpolarized cells secreted significantly more TGF-β2 supporting the contention that loss of polarity of RPE in PVR leads to rise of intravitreal TGF-β2. Active TGF-β2, secreted mainly from apical side of polarized RPE, represented 6–10% of total TGF-β2. In conclusion, polarity is an important determinant of TGF-β2 secretion in RPE. Low levels of apically secreted active TGF-β2 may play a role in the normal physiology of the subretinal space. Comparable secretion of TGF-β from polarized hESC-RPE and hRPE supports the potential for hESC-RPE in RPE replacement therapies.