Azeddine Atfi

Evidence for a Role of Rho-like GTPases and Stress-activated Protein Kinase/c-Jun N-terminal Kinase (SAPK/JNK) in Transforming Growth Factor β-mediated Signaling

Transforming growth factor β (TGF-β) is a multifunctional factor that induces a wide variety of cellular processes which affect growth and differentiation. TGF-β exerts its effects through a heteromeric complex between two transmembrane serine/threonine kinase receptors, the type I and type II receptors. However, the intracellular signaling pathways through which TGF-β receptors act to generate cellular responses remain largely undefined. Here, we report that TGF-β initiates a signaling cascade leading to stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) activation. Expression of dominant-interfering forms of various components of the SAPK/JNK signaling pathways including Rho-like GTPases, mitogen-activated protein kinase (MAPK) kinase kinase 1 (MEKK1), MAPK kinase 4 (MKK4), SAPK/JNK, and c-Jun abolishes TGF-β-mediated signaling. Therefore, the SAPK/JNK activation contributes to TGF-β signaling.

Activation of mitogen-activated protein kinase cascades is involved in regulation of bone morphogenetic protein-2-induced osteoblast differentiation in pluripotent C2C12 cells

Bone morphogenetic protein (BMP)-2, a member of the transforming growth factor-beta (TGF-beta) superfamily, is able to induce osteoblastic differentiation of C2C12 cells. Both Smad and mitogen-activated protein kinase (MAPK) pathways are essential components of the TGF-beta superfamily signaling machinery. Although Smads have been demonstrated to participate in the BMP-2-induced osteoblastic differentiation of C2C12 cells, the role of MAPK has not been addressed. This report shows that BMP-2 activates ERK and p38, but not JNK, in C2C12 cells. Pretreatment of cells with the p38 inhibitor, SB203580, dramatically reduced BMP-2-induced expression of the osteoblast markers alkaline phosphatase (ALP) and osteocalcin (OC). Nevertheless, overexpression of MKK3, a protein kinase that phosphorylates and activates p38, failed to induce ALP or OC expression in the absence of BMP-2, indicating that p38 activation is necessary but not sufficient for the acquisition of the osteoblast phenotype by these cells. Although ALP induction was increased slightly in the presence of PD-98059, a selective inhibitor of the ERK cascade, this compound significantly inhibited both steady-state and BMP-2-induced OC RNA levels. Our results indicate that p38 and ERK cascades play a crucial role in the osteoblast differentiation of C2C12 cells mediated by BMP-2.

Induction of apoptosis by DPC4, a transcriptional factor regulated by transforming growth factor-beta through stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) signaling pathway.

Many of the actions of serine/threonine kinase receptors for the transforming growth factor-β (TGFβ) are mediated by DPC4, a human MAD-related protein identified as a tumor suppressor gene in pancreatic carcinoma. Overexpression of DPC4 is sufficient to induce the activation of gene expression and cell cycle arrest, characteristic of the TGFβ response. The stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) is also one of the downstream targets required for TGFβ-mediated signaling. Here we report that expression of the dominant-interfering mutant of various components of the SAPK/JNK cascade specifically blocked both TGFβ and DPC4-induced gene expression. These dominant-interfering mutants also inhibited TGFβ-stimulated DPC4 transcriptional activity. Moreover, we find that overexpression of DPC4 causes transfected cells to undergo the morphological changes typical of apoptosis. These findings define a mechanism whereby TGFβ signals mediated by DPC4 and SAPK/JNK cascade are integrated in the nucleus to activate gene expression and identify a new cellular function for DPC4.

Tumor Necrosis Factor-α Inhibits Transforming Growth Factor-β /Smad Signaling in Human Dermal Fibroblasts via AP-1 Activation

Understanding the molecular mechanisms underlying the antagonistic activities of tumor necrosis factor-α (TNF-α) against transforming growth factor-β (TGF-β) is of utmost importance given the physiopathological implications of these cytokines. In this report, we demonstrate that TNF-α prevents TGF-β-induced Smad-specific gene transactivation without inducing detectable levels of inhibitory Smad7 in human dermal fibroblasts. On the other hand, c-Jun and JunB, both induced by TNF-α, block Smad3-mediated transcription. Expression of antisense c-Jun mRNA prevents TNF-α inhibition of TGF-β/Smad signaling whereas that of dominant-negative Iκ-B kinase-α or antisense Smad7 does not. We provide evidence for off-DNA interactions between Smad3 and both c-Jun and JunB accompanied with reduced Smad3-DNA interactions. Finally, we show that overexpression of the transcriptional co-activator p300 prevents TNF-α/AP-1 inhibition of TGF-β/Smad signaling. These data suggest that TNF-α interferes with Smad signaling through the induction of AP-1 components, the latter forming off-DNA complexes with Smad3 and preventing its binding to specificcis-element(s). In addition, Jun members compete with Smad3 for the common transcription co-activator p300. These two mechanisms are likely to act in concert to decrease Smad-specific transcription.

Yes-associated protein (YAP65) interacts with Smad7 and potentiates its inhibitory activity against TGF-beta/Smad signaling.

Members of the TGF-β family of growth factors signal from the cell surface through serine/threonine kinase receptors. Intracellular propagation of the signal occurs by phosphorylation of intracellular proteins of the Smad family. Smad7 belongs to the subclass of inhibitory Smads that function as antagonists of TGF-β signaling. A yeast two-hybrid screen of a human placental cDNA expression library using full-length mouse Smad7 as bait identified Yes-Associated Protein (YAP65) as a novel Smad7-interacting protein. The association of Smad7 with YAP65 was confirmed using co-expressed tagged proteins in COS-7 cells. Deletion of the PY motif of Smad7 reduced but did not abolish YAP65-Smad7 association, suggesting the existence of several interacting domains. We demonstrate that YAP65 potentiates the inhibitory activity of Smad7 against TGF-β-induced, Smad3/4-dependent, gene transactivation. Furthermore, YAP65 augments the association of Smad7 to activated TGF-β receptor type I (TβRI), whereas YAP65(1–301), which exerts a dominant-negative effect against Smad7-driven inhibition of TGF-β signaling, reduces these interactions. Together, these data provide the first evidence that YAP65 is a Smad7 partner that facilitates the recruitment of the latter to activated TβRI, and enhances the inhibitory activity of Smad7 against TGF-β signaling.

The novel E3 ubiquitin ligase Tiul1 associates with TGIF to target Smad2 for degradation

Ubiquitin‐dependent degradation plays an important role in the negative regulation of TGF‐β signaling. Here, we identify Tiul1 (for TGIF interacting ubiquitin ligase 1), a novel E3 ubiquitin ligase that inhibits TGF‐β signaling by targeting both the activated receptor and Smad2 for degradation. Tiul1 associates constitutively with Smad7 and induces degradation of the activated type I receptor without affecting the expression levels of Smad7. Tiul1 can also interact with Smad2 and the nuclear corepressor TGIF upon activation of TGF‐β signaling. Like Smad7, the steady‐state levels of TGIF are not affected by Tiul1, but the interaction of Tiul1 with TGIF allows this ubiquitin ligase to target Smad2 for degradation. Consistent with this, overexpression of Tiul1 suppressed TGF‐β‐induced growth arrest and transcriptional responses. In addition, silencing of Tiul1 or TGIF genes by siRNA resulted in suppression of the TGF‐β‐dependent degradation of Smad2 and an enhancement of TGF‐β‐mediated gene expression. These results reveal a new role for TGIF as a component of a ubiquitin ligase complex that mediates the degradation of Smad2 in response to TGF‐β signaling.

Smad7 inhibits the survival nuclear factor κB and potentiates apoptosis in epithelial cells

In this study, we examined the effect of the stable expression of Smad7 in two different cell lines on apoptosis induced by various stimuli including TGF-β, serum withdrawal, loss of cell adhesion (anoikis) and TNF-α. Smad7 increased TGF-β-mediated apoptosis in Mv1Lu cells as well as anoikis and/or serum withdrawal-induced apoptosis in Mv1Lu and MDCK cells. Smad7 markedly decreased the activity of the survival NF-κB transcription factor in MDCK cells. Interestingly, the stable expression of oncogenic Ras in MDCK cells which suppressed Smad7 inhibition of NF-κB also suppressed Smad7 potentiation of serum withdrawal-induced apoptosis and anoikis. In addition, Smad7 inhibited TNF-α stimulation of NF-κB and increased TNF-α-mediated apoptosis in MDCK cells. Our results provide the first evidence that Smad7 induces sensitization of cells to different forms of cell death. They moreover demonstrate that Smad7 inhibits the survival NF-κB factor, providing a potential mechanism whereby Smad7 potentiates cell death.

Liver cancer-derived hepatitis C virus core proteins shift TGF-beta responses from tumor suppression to epithelial-mesenchymal transition.

Background Chronic hepatitis C virus (HCV) infection and associated liver cirrhosis represent a major risk factor for hepatocellular carcinoma (HCC) development. TGF-β is an important driver of liver fibrogenesis and cancer; however, its actual impact in human cancer progression is still poorly known. The aim of this study was to investigate the role of HCC-derived HCV core natural variants on cancer progression through their impact on TGF-β signaling. Principal Findings We provide evidence that HCC-derived core protein expression in primary human or mouse hepatocyte alleviates TGF-β responses in terms or growth inhibition or apoptosis. Instead, in these hepatocytes TGF-β was still able to induce an epithelial to mesenchymal transition (EMT), a process that contributes to the promotion of cell invasion and metastasis. Moreover, we demonstrate that different thresholds of Smad3 activation dictate the TGF-β responses in hepatic cells and that HCV core protein, by decreasing Smad3 activation, may switch TGF-β growth inhibitory effects to tumor promoting responses. Conclusion/Significance Our data illustrate the capacity of hepatocytes to develop EMT and plasticity under TGF-β, emphasize the role of HCV core protein in the dynamic of these effects and provide evidence for a paradigm whereby a viral protein implicated in oncogenesis is capable to shift TGF-β responses from cytostatic effects to EMT development.

c-Jun interacts with the corepressor TG-interacting factor (TGIF) to suppress Smad2 transcriptional activity.

The Sma and Mad related (Smad) family proteins are critical mediators of the transforming growth factor-β (TGF-β) superfamily signaling. After TGF-β-mediated phosphorylation and association with Smad4, Smad2 moves to the nucleus and activates expression of specific genes through cooperative interactions with DNA-binding proteins, including members of the winged-helix family of transcription factors, forkhead activin signal transducer (FAST)-1 and FAST2. TGF-β has also been described to activate other signaling pathways, such as the c-Jun N-terminal Kinase (JNK) pathway. Here, we show that activation of JNK cascade blocked the ability of Smad2 to mediate TGF-β-dependent activation of the FAST proteins. This inhibitory activity is mediated through the transcriptional factor c-Jun, which enhances the association of Smad2 with the nuclear transcriptional corepressor TG-interacting factor (TGIF), thereby interfering with the assembly of Smad2 and the coactivator p300 in response to TGF-β signaling. Interestingly, c-Jun directly binds to the nuclear transcriptional corepressor TGIF and is required for TGIF-mediated repression of Smad2 transcriptional activity. These studies thus reveal a mechanism for suppression of Smad2 signaling pathway by JNK cascade through transcriptional repression.

Zfp521 controls bone mass by HDAC3-dependent attenuation of Runx2 activity

Zfp521 regulates osteoblast development during lineage commitment and osteoblast maturation by suppressing Runx2 transcriptional activity.