Transcriptional induction of Smurf2 ubiquitin ligase by TGF-beta

Smad ubiquitination regulatory factor 2 (Smurf2), a ubiquitin ligase for Smads, plays critical roles in the regulation of transforming growth factor-beta (TGF-beta)-Smad signaling via ubiquitin-dependent degradation of Smad2 and Smad7. We found that TGF-beta stimulates Smurf2 expression. TGF-beta activated the Smurf2 promoter in a TGF-beta responsive cell lines, whereas IL-1alpha, PDGF and epidermal growth factor did not. TGF-beta-mediated Smurf2 promoter activation was inhibited by Smad7 or an activin receptor-like kinase 5 inhibitor but not by dominant negative Smad or disruption of Smad-binding elements in the promoter. Moreover, inhibition of the phosphatidil inositol 3 kinase (PI3K)/Akt pathway suppressed TGF-beta-mediated Smurf2 induction. These results suggest that TGF-beta stimulates Smurf2 expression by Smad-independent pathway such as PI3K/Akt pathway via TGF-beta receptor.     

Angiotensin II-induced differentiation of adipose tissue-derived mesenchymal stem cells to smooth muscle-like cells

Angiotensin II (Ang II) is involved in the development of cardiovascular disease and vascular remodeling. In this study, we demonstrate that treatment of human adipose tissue-derived mesenchymal stem cells (hADSCs) with Ang II increased the expression of smooth muscle-specific genes, including alpha-smooth muscle actin (alpha-SMA), calponin, h-caldesmon, and smooth muscle myosin heavy chain (SM-MHC), and also elicited the secretion of transforming growth factor-beta1 (TGF-beta1) and delayed phosphorylation of Smad2. The Ang II-induced expression of alpha-SMA and delayed phosphorylation of Smad2 were blocked by pretreatment of the cells with a TGF-beta type I receptor kinase inhibitor, SB-431542, small interference RNA-mediated depletion of endogenous Smad2, and adenoviral expression of Smad7. Furthermore, the Ang II-induced TGF-beta1 secretion, alpha-SMA expression, and delayed phosphorylation of Smad2 in hADSCs were abrogated by the MEK inhibitor U0126, suggesting a pivotal role of MEK/ERK pathway in the Ang II-induced activation of TGF-beta1-Smad2 signaling pathway. The smooth muscle-like cells which were differentiated from hADSCs by Ang II treatment exhibited contraction in response to 60mM KCl. These results suggest that Ang II induces differentiation of hADSCs to contractile smooth muscle-like cells through ERK-dependent activation of the autocrine TGF-beta1-Smad2 crosstalk pathway.     

LIM-kinase 2 and cofilin phosphorylation mediate actin cytoskeleton reorganization induced by transforming growth factor-beta

Reorganization of the actin cytoskeleton in response to growth factor signaling, such as transforming growth factor beta (TGF-beta), controls cell adhesion, motility, and growth of diverse cell types. In Swiss3T3 fibroblasts, a widely used model for studies of actin reorganization, TGF-beta1 induced rapid actin polymerization into stress fibers and concomitantly activated RhoA and RhoB small GTPases. Consequently, dominant-negative RhoA and RhoB mutants blocked TGF-beta1-induced actin reorganization. Because Rho GTPases are known to regulate the activity of LIM-kinases (LIMK), we found that TGF-beta1 induced LIMK2 phosphorylation with similar kinetics to Rho activation. Cofilin and LIMK2 co-precipitated and cofilin became phosphorylated in response to TGF-beta1, whereas RNA interference against LIMK2 blocked formation of new stress fibers by TGF-beta1. Because the kinase ROCK1 links Rho GTPases to LIMK2, we found that inhibiting ROCK1 activity blocked completely TGF-beta1-induced LIMK2/cofilin phosphorylation and downstream stress fiber formation. We then tested whether the canonical TGF-beta receptor/Smad pathway mediates regulation of the above effectors and actin reorganization. Adenoviruses expressing constitutively activated TGF-beta type I receptor led to robust actin reorganization and Rho activation, whereas the constitutively activated TGF-beta type I receptor with mutated Smad docking sites (L45 loop) did not affect either actin organization or Rho activity. In line with this, ectopic expression of the inhibitory Smad7 inhibited TGF-beta1-induced Rho activation and cytoskeletal reorganization. Our data define a novel pathway emanating from the TGF-beta type I receptor and leading to regulation of actin assembly, via the kinase LIMK2.     

Smad proteins exist as monomers in vivo and undergo homo- and hetero-oligomerization upon activation by serine/threonine kinase receptors.

Smad proteins are signal transducers for the members of the transforming growth factor-beta (TGF-beta) superfamily. Here we show that, in the absence TGF-beta stimulation, Smads exist as monomers in vivo. Smad2 and Smad3 form homo-oligomers upon phosphorylation by the constitutively active TGF-beta type I receptor, and this oligomerization does not require Smad4. Major portions of Smad4, Smad6 and Smad7 are also present as monomers in vivo. Analysis using a cross-linking reagent suggested that the Smad2 oligomer induced by receptor activation is a trimer. Studies by gel chromatography demonstrated that the Smad2-Smad4 heteromer is not larger than the Smad2 homomer. Moreover, overexpression of Smad4 prevented Smad2 from forming a homo-oligomer. These findings suggest that Smad2 may form a homotrimer, or heterotrimers with Smad4, which are probably composed of two and one, or one and two molecules of Smad2 and Smad4, respectively, depending on the amount of each protein. Gel-mobility shift assay revealed that the Smad3 homomer and Smad3-Smad4 heteromer constitute DNA-binding complexes. Transition of the Smad proteins from monomers to oligomers is thus a critical event in the signal transduction of the TGF-beta superfamily members.     

Transforming growth factor-beta 1 inhibition of macrophage activation is mediated via Smad3

Activated macrophages are critical cellular participants in inflammatory disease states. Transforming growth factor (TGF)-beta1 is a growth factor with pleiotropic effects including inhibition of immune cell activation. Although the pathway of gene activation by TGF-beta1 via Smad proteins has recently been elucidated, suppression of gene expression by TGF-beta1 remains poorly understood. We found that of Smad1-Smad7, Smad3 alone was able to inhibit expression of markers of macrophage activation (inducible nitric-oxide synthase and matrix metalloproteinase-12) following lipopolysaccharide treatment in gene reporter assays. Transient and constitutive overexpression of a dominant negative Smad3 opposed the inhibitory effect of TGF-beta1. Domain swapping experiments suggest that both the Smad MH-1 and MH-2 domains are required for inhibition. Mutation of a critical amino acid residue required for DNA binding in the MH-1 of Smad3 (R74A) resulted in the loss of inhibition. Transient overexpression of p300, an interactor of the Smad MH-2 domain, partially alleviated the inhibition by TGF-beta1/Smad3, suggesting that inhibition of gene expression may be due to increased competition for limiting amounts of this coactivator. Our results have implications for the understanding of gene suppression by TGF-beta1 and for the regulation of activated macrophages by TGF-beta1.     

Sphingosylphosphorylcholine stimulates expression of fibronectin through TGF-beta1-Smad-dependent mechanism in human mesenchymal stem cells

Sphingosylphosphorylcholine (SPC) has been reported to stimulate the expression of fibronectin (FN), which plays a key role in cell recruitment and adhesion during wound healing. In a previous study, we reported that SPC induces differentiation of human adipose tissue-derived mesenchymal stem cells (hATSCs) to smooth muscle-like cell types through ERK-dependent autocrine secretion of TGF-beta1 and delayed activation of the TGF-beta1-Smad pathway. In the present study, we demonstrated that SPC dose- and time-dependently increased the expression of FN in hATSCs. Pretreatment of the cells with U0126, an MEK inhibitor, markedly attenuated the SPC-induced expression of FN and delayed phosphorylation of Smad2, suggesting that ERK is involved in the SPC induction of FN expression through activation of Smad2. In addition, the SPC-induced expression of FN and delayed activation of Smad2 were abrogated by SB-431542, a TGF-beta type I receptor kinase inhibitor, or anti-TGF-beta1 neutralizing antibody. Furthermore, the SPC-induced expression of FN was abrogated by adenoviral expression of Smad7, an inhibitory Smad, or short interference RNA (siRNA)-mediated depletion of endogenous Smad2 expression, suggesting that SPC induces the expression of FN through ERK-dependent activation of the TGF-beta1-Smad2 crosstalk pathway. Adhesion of U937 monocytic cells to hATSCs was enhanced by pretreatment of hATSCs with SPC or TGF-beta1 for 4 days, and the peptide GRGDSP (an antagonist of fibronectin receptors) blocked the adhesion of U937 cells to the hATSCs. These results led us to suggest that SPC-induced FN expression plays a pivotal role in the wound healing by stimulating adhesion and recruitment of leukocytes.     

Transforming growth factor beta activation of c-Abl is independent of receptor internalization and regulated by phosphatidylinositol 3-kinase and PAK2 in mesenchymal cultures

Transforming growth factor beta (TGF-beta) modulates a number of cellular phenotypes as divergent as growth stimulation and growth inhibition. Although the Smad pathway is critical for many of these responses, recent evidence indicates that Smad-independent pathways may also have a critical role. One such protein previously shown to regulate TGF-beta action independent of the Smad proteins is the c-Abl nonreceptor tyrosine kinase. In the current study we determined that TGF-beta receptor signaling activates c-Abl kinase activity in a subset of fibroblast but not epithelial cultures. This cell type-specific response occurs in a membrane-proximal locale independent of receptor internalization and upstream of dynamin action. Although c-Abl activation by TGF-beta is independent of Smad2 or Smad3, it is prevented by inhibitors of phosphatidylinositol 3-kinase or PAK2. Thus, c-Abl represents a target downstream of phosphatidylinositol 3-kinase-activated PAK2, which differentiates TGF-beta signaling in fibroblasts and epithelial cell lines and integrates serine/threonine receptor kinases with tyrosine kinase pathways.     

A novel signaling pathway of ADP-ribosyl cyclase activation by angiotensin II in adult rat cardiomyocytes

ADP-ribosyl cyclase (ADPR-cyclase) produces a Ca(2+)-mobilizing second messenger, cADP-ribose (cADPR), from NAD(+). In this study, we investigated the molecular basis of ADPR-cyclase activation in the ANG II signaling pathway and cellular responses in adult rat cardiomyocytes. The results showed that ANG II generated biphasic intracellular Ca(2+) concentration increases that include a rapid transient Ca(2+) elevation via inositol trisphosphate (IP(3)) receptor and sustained Ca(2+) rise via the activation of L-type Ca(2+) channel and opening of ryanodine receptor. ANG II-induced sustained Ca(2+) rise was blocked by a cADPR antagonistic analog, 8-bromo-cADPR, indicating that sustained Ca(2+) rise is mediated by cADPR. Supporting the notion, ADPR-cyclase activity and cADPR production by ANG II were increased in a time-dependent manner. Application of pharmacological inhibitors and immunological analyses revealed that cADPR formation was activated by sequential activation of Src, phosphatidylinositol 3-kinase (PI 3-kinase)/protein kinase B (Akt), phospholipase C (PLC)-gamma1, and IP(3)-mediated Ca(2+) signal. Inhibitors of these signaling molecules not only completely abolished the ANG II-induced Ca(2+) signals but also inhibited cADPR formation. Application of the cADPR antagonist and inhibitors of upstream signaling molecules of ADPR-cyclase inhibited ANG II-stimulated hypertrophic responses, which include nuclear translocation of Ca(2+)/calcineurin-dependent nuclear factor of activated T cells 3, protein expression of transforming growth factor-beta1, and incorporation of [(3)H]leucine in cardiomyocytes. Taken together, these findings suggest that activation of ADPR-cyclase by ANG II entails a novel signaling pathway involving sequential activation of Src, PI 3-kinase/Akt, and PLC-gamma1/IP(3) and that the activation of ADPR-cyclase can lead to cardiac hypertrophy.