Rainer Heuchel

Profibrogenic transforming growth factor‐β/activin receptor–like kinase 5 signaling via connective tissue growth factor expression in hepatocytes

Connective tissue growth factor (CTGF) is important for transforming growth factor‐β (TGF‐β)–induced liver fibrogenesis. Hepatic stellate cells have been recognized as its major cellular source in the liver. Here we demonstrate the induction of CTGF expression in hepatocytes of damaged livers and identify a molecular mechanism responsible for it. CTGF expression was found by immunohistochemistry in bile duct epithelial cells, hepatic stellate cells, and hepatocytes in fibrotic liver tissue from patients with chronic hepatitis B infection. Similarly, CTGF expression was induced in hepatocytes of carbon tetrachloride–treated mice. CTGF expression and secretion were detected spontaneously in a medium of hepatocytes after 3 days of culture, which was enhanced by stimulation with TGF‐β. TGF‐β–induced CTGF expression was mediated through the activin receptor–like kinase 5 (ALK5)/Smad3 pathway, whereas activin receptor–like kinase 1 activation antagonized this effect. CTGF expression in the liver tissue of TGF‐β transgenic mice correlated with serum TGF‐β levels. Smad7 overexpression in cultured hepatocytes abrogated TGF‐β–dependent and intrinsic CTGF expression, indicating that TGF‐β signaling was required. In line with these data, hepatocyte‐specific transgenic Smad7 reduced CTGF expression in carbon tetrachloride–treated animals, whereas in Smad7 knockout mice, it was enhanced. Furthermore, an interferon gamma treatment of patients with chronic hepatitis B virus infection induced Smad7 expression in hepatocytes, leading to decreased CTGF expression and fibrogenesis. Conclusion: Our data provide evidence for the profibrogenic activity of TGF‐β directed to hepatocytes and mediated via the up‐regulation of CTGF. We identify ALK5‐dependent Smad3 signaling as the responsible pathway inducing CTGF expression, which can be hindered by an activated activin receptor–like kinase 1 pathway and completely inhibited by TGF‐β antagonist Smad7. (HEPATOLOGY 2007.)

Deletion of Exon I of SMAD7 in Mice Results in Altered B Cell Responses

The members of the TGF-β superfamily, i.e., TGF-β isoforms, activins, and bone morphogenetic proteins, regulate growth, differentiation, and apoptosis, both during embryonic development and during postnatal life. Smad7 is induced by the TGF-β superfamily members and negatively modulates their signaling, thus acting in a negative, autocrine feedback manner. In addition, Smad7 is induced by other stimuli. Thus, it can fine-tune and integrate TGF-β signaling with other signaling pathways. To investigate the functional role(s) of Smad7 in vivo, we generated mice deficient in exon I of Smad7, leading to a partial loss of Smad7 function. Mutant animals are viable, but significantly smaller on the outbred CD-1 mouse strain background. Mutant B cells showed an overactive TGF-β signaling measured as increase of phosphorylated Smad2-positive B cells compared with B cells from wild-type mice. In agreement with this expected increase in TGF-β signaling, several changes in B cell responses were observed. Mutant B cells exhibited increased Ig class switch recombination to IgA, significantly enhanced spontaneous apoptosis in B cells, and a markedly reduced proliferative response to LPS stimulation. Interestingly, LPS treatment reverted the apoptotic phenotype in the mutant cells. Taken together, the observed phenotype highlights a prominent role for Smad7 in development and in regulating the immune system’s response to TGF-β.

Disruption of the Smad7 gene enhances CCI4-dependent liver damage and fibrogenesis in mice

Transforming growth factor‐β (TGF‐β) signalling is induced in liver as a consequence of damage and contributes to wound healing with transient activation, whereas it mediates fibrogenesis with long‐term up‐regulation in chronic disease. Smad‐dependent TGF‐β effects are blunted by antagonistic Smad7, which is transcriptionally activated as an immediate early response upon initiation of TGF‐β signalling in most cell types, thereby providing negative feedback regulation. Smad7 can be induced by other cytokines, e.g. IFN‐γ, leading to a crosstalk of these signalling pathways. Here we report on a novel mouse strain, denoted S7ΔE1, with a deletion of exon I from the endogenous smad7 gene. The mice were viable and exhibited normal adult liver architecture. To obtain insight into Smad7‐depend‐ent protective effects, chronic liver damage was induced in mice by carbon tetrachloride (CCI4) administration. Subsequent treatment, elevated serum liver enzymes indicated enhanced liver damage in mice lacking functional Smad7. CCI4‐dependent Smad2 phosphoryla‐tion was pronounced in S7ΔE1 mice and accompanied by increased numbers of α‐smooth muscle actin positive ‘activated’ HSCs. There was evidence for matrix accumulation, with elevated collagen deposition as assessed morphometrically in Sirius red stained tissue and confirmed with higher levels of hydroxyproline in S7ΔE1 mice. In addition, the number of CD43 positive infiltrating lymphocytes as well as of apoptotic hepatocytes was increased. Studies with primary hepatocytes from S7ΔE1 and wild‐type mice indicate that in the absence of functional Smad7 protein, hepatocytes are more sensitive for TGF‐β effects resulting in enhanced cell death. Furthermore, S7ΔE1 hepatocytes display increased oxidative stress and cell damage in response to CCI4, as measured by reactive oxygen species production, glutathione depletion, lactate dehydrogenase release and lipid peroxidation. Using an ALK‐5 inhibitor all investigated CCI4 effects on hepatocytes were blunted, confirming participation of TGF‐β signalling. We conclude that Smad7 mediates a protective effect from adverse TGF‐β signalling in damaged liver, re‐iterating its negative regulatory loop on signalling.

A gain of function mutation in the activation loop of platelet-derived growth factor beta-receptor deregulates its kinase activity.

The platelet-derived growth factor receptors (PDGFRs) are receptor tyrosine kinases implicated in multiple aspects of cell growth, differentiation, and survival. Recently, a gain of function mutation in the activation loop of the human PDGFRα has been found in patients with gastrointestinal stromal tumors. Here we show that a mutation in the corresponding codon in the activation loop of the murine PDGFRβ, namely an exchange of asparagine for aspartic acid at amino acid position 849 (D849N), confers transforming characteristics to embryonic fibroblasts from mutant mice, generated by a knock-in strategy. By comparing the enzymatic properties of the wild-type versus the mutant receptor protein, we demonstrate that the D849N mutation lowers the threshold for kinase activation, causes a dramatic alteration in the pattern of tyrosine phosphorylation kinetics following ligand stimulation, and induces a ligand-independent phosphorylation of several tyrosine residues. These changes result in deregulated recruitment of specific signal transducers. The GTPase-activating protein for Ras (RasGAP), a negative regulator of the Ras mitogenic pathway, displayed a delayed binding to the mutant receptor. Moreover, we have observed enhanced ligand-independent ERK1/2 activation and an increased proliferation of mutant cells. The p85 regulatory subunit of the phosphatidylinositol 3 ′-kinase was constitutively associated with the mutant receptor, and this ligand-independent activation of the phosphatidylinositol 3′-kinase pathway may explain the observed strong protection against apoptosis and increased motility in cellular wounding assays. Our findings support a model whereby an activating point mutation results in a deregulated PDGFRβ with oncogenic predisposition.

The human α11 integrin promoter drives fibroblast-restricted expression in vivo and is regulated by TGF-β1 in a Smad- and Sp1-dependent manner

Integrin alpha11beta1 is expressed by ectomesenchymally- and mesodermally-derived fibroblasts and is the major collagen receptor on embryonic fibroblasts. We have previously characterized a 3kb human alpha11 promoter region in vitro. In the current study we generated promoter-LacZ reporter transgenic mice to examine the ability of the 3kb alpha11 promoter to drive tissue-specific expression also in vivo. Our data show that the 3 kb alpha11 promoter contains most of the regulatory elements that direct ectomesenchymal and mesodermal fibroblast-specific expression. Not much is known about integrin alpha11 regulation by TGF-beta family members and the potential role of alpha11 in TGF-beta1 driven processes such as fibrosis and wound contraction. In the current study we show that TGF-beta1 induces alpha11 transcription in the fibrosarcoma cell line HT1080 as well as in primary fibroblasts. Co-transfection of an expression plasmid encoding constitutively active ALK5 together with alpha11 promoter-luciferase reporter constructs demonstrated that TGF-beta1 responsive elements are located within the 3kb alpha11 promoter. Serial deletions located TGF-beta1 responsiveness to the proximal promoter (nt -176/+25) as well as to the region extending to nt -330. Transfection and expression of the inhibitory Smad7 in the cells attenuated the TGF-beta1-dependent alpha11 induction both at the RNA and the protein level. Mutation and deletion analyses identified a Smad-binding element, SBE2 (nt -182/-176), as an important Smad3-binding site in this part of the promoter. Further analyses suggested that the Sp1-binding site SBS1 (nt -140/-134) takes part in the responsiveness to TGF-beta1 in a Smad2-dependent manner. In summary, our data confirm that 3kb of the alpha11 promoter is efficient in driving tissue-specific expression in vivo. We also demonstrate that this promoter confers TGF-beta1 responsiveness which appears to rely on both a Smad-binding element at nt -182/-176 and a Sp1-binding site at nt -140/-134. Our data furthermore indicate that additional elements needed for TGF-beta1 responsiveness are located upstream in the -2962/-330 promoter region.

Platelet-derived growth factor receptor-β, carrying the activating mutation D849N, accelerates the establishment of B16 melanoma

BackgroundPlatelet-derived growth factor (PDGF)-BB and PDGF receptor (PDGFR)-β are mainly expressed in the developing vasculature, where PDGF-BB is produced by endothelial cells and PDGFR-β is expressed by mural cells, including pericytes. PDGF-BB is produced by most types of solid tumors, and PDGF receptor signaling participates in various processes, including autocrine stimulation of tumor cell growth, recruitment of tumor stroma fibroblasts, and stimulation of tumor angiogenesis. Furthermore, PDGF-BB-producing tumors are characterized by increased pericyte abundance and accelerated tumor growth. Thus, there is a growing interest in the development of tumor treatment strategies by blocking PDGF/PDGFR function. We have recently generated a mouse model carrying an activated PDGFR-β by replacing the highly conserved aspartic acid residue (D) 849 in the activating loop with asparagine (N). This allowed us to investigate, in an orthotopic tumor model, the role of increased stromal PDGFR-β signaling in tumor-stroma interactions.MethodsB16 melanoma cells lacking PDGFR-β expression and either mock-transfected or engineered to express PDGF-BB, were injected alone or in combination with matrigel into mice carrying the activated PDGFR-β (D849N) and into wild type mice. The tumor growth rate was followed and the vessel status of tumors, i.e. total vessel area/tumor, average vessel surface and pericyte density of vessels, was analyzed after resection.ResultsTumors grown in mice carrying an activated PDGFR-β were established earlier than those in wild-type mice. In this early phase, the total vessel area and the average vessel surface were higher in tumors grown in mice carrying the activated PDGFR-β (D849N) compared to wild-type mice, whereas we did not find a significant difference in the number of tumor vessels and the pericyte abundance around tumor vessels between wild type and mutant mice. At later phases of tumor progression, no significant difference in tumor growth rate was observed between wild type mice and mutant mice, although the pericyte coverage was higher around tumor vessels from mutant mice.ConclusionOur findings suggest that the activated PDGFR-β (D849N) in the host animal increased the total vessel area and the average vessel surface even in PDGF-negative tumors, resulting in a shorter lag phase during tumor establishment.

A gain-of-function mutation in the PDGFR-β alters the kinetics of injury response in liver and skin

Platelet-derived growth factor (PDGF) isoforms stimulate cell proliferation, migration and survival. We recently generated mice carrying a gain-of-function mutation within the activation loop of PDGF β-receptor (PDGFR-β D849N). Embryonic fibroblasts derived from these mice show elevated basal phosphorylation and altered kinetics for ligand-induced activation of PDGFR-β, as well as enhanced proliferation and migration. To investigate the effect of this mutation in vivo, we used carbon tetrachloride-induced liver injury as a model system. We observed a higher basal activation of mutant PDGFR-β in unchallenged livers; however, the difference in activation upon carbon tetrachloride stimulation was lower than expected, an effect that might be explained by a delayed response of the mutated receptor toward reactive oxygen species. Mutant mice showed enhanced proliferation of nonparenchymal liver cells and activation of hepatic stellate cells, leading to a small increase in early fibrosis formation. Another mouse strain lacking the binding site for phosphatidylinositol-3′ kinase in PDGFR-β showed the reverse phenotype. These results suggest an important role for PDGFR-β signaling in the early injury-response. We confirmed this hypothesis with a second injury model, cutaneous wound healing, where we observed earlier proliferation and formation of granulation tissue in D849N-mutant mice.