Novel Regulators of the TGF-β Signaling Pathway

The transforming growth factor-β (TGF-β) superfamily consists of related multifunctional cytokines, which include TGF-βs, activins, and bone morphogenetic proteins (BMPs) and coordinate several biological responses in diverse cell types. The biological activity of TGF-β members is executed by transmembrane serine/threonine kinase receptors and intracellular Smad proteins. The effects of TGF-β on the epithelium are of high interest. Carcinomas (tumors of epithelial origin) are the most common type of human cancer and frequently exhibit aberrant responses to TGF-β. Therefore, TGF-β can be defined as tumor suppressor as it inhibits growth of normal epithelial cells. However, TGF-β also induces an epithelial-mesenchymal transition (EMT), a key component of metastasis, and thus promotes cancer spread. The scope of this thesis is the mechanism of TGF-β signaling in epithelial cells. We established that only TGF-β, but not BMP pathways can elicit EMT. Moreover, we found that Smad signaling is critical for regulation of EMT. In a transcriptomic analysis, we identified a large group of novel genes, whose regulation is pivotal for TGF-β-induced EMT and metastasis. We focused on two of such genes, Id2 and Id3. Interestingly, we found that TGF-β-induced repression of Ids is necessary for inducing EMT and potent cell cycle arrest. BMP increases expression of Ids and therefore it cannot induce the same biological responses as TGF-β. Hence, knock-down of endogenous Id2 and Id3 proteins sensitized epithelial cell to BMP-7. We proposed a model, in which Id2 and Id3 are important components controlling concerted regulation of cell proliferation and EMT downstream of TGF-β pathways. Furthermore, we identified a serine/threonine kinase, SNF1LK, whose mRNA is rapidly induced by TGF-β in epithelial cells. We found that SNF1LK is a negative regulator of the TGF-β pathway and it promotes TGF-β receptor turnover. Subsequently, we demonstrated that SNF1LK together with Smad7 and Smurf2 targets TGF-β receptor for ubiquitin-dependent degradation. Furthermore, SNF1LK interacts with proteasomes, suggesting that SNF1LK serves as bridge between ubiquitinated receptors and proteasomes, helping proteasomes to recognize the ubiquitinated cargo destined for degradation. We therefore established a novel negative feedback regulatory mechanism of TGF-β signaling

TGF-β and the Smad Signaling Pathway Support Transcriptomic Reprogramming during Epithelial-Mesenchymal Cell Transition

Epithelial-mesenchymal transition (EMT) contributes to normal tissue patterning and carcinoma invasiveness. We show that transforming growth factor (TGF)-β/activin members, but not bone morphogenetic protein (BMP) members, can induce EMT in normal human and mouse epithelial cells. EMT correlates with the ability of these ligands to induce growth arrest. Ectopic expression of all type I receptors of the TGF-β superfamily establishes that TGF-β but not BMP pathways can elicit EMT. Ectopic Smad2 or Smad3 together with Smad4 enhanced, whereas dominant-negative forms of Smad2, Smad3, or Smad4, and wild-type inhibitory Smad7, blocked TGF-β–induced EMT. Transcriptomic analysis of EMT kinetics identified novel TGF-β target genes with ligand-specific responses. Using a TGF-β type I receptor that cannot activate Smads nor induce EMT, we found that Smad signaling is critical for regulation of all tested gene targets during EMT. One such gene, Id2, whose expression is repressed by TGF-β1 but induced by BMP-7 is critical for regulation of at least one important myoepithelial marker, α-smooth muscle actin, during EMT. Thus, based on ligand-specific responsiveness and evolutionary conservation of the gene expression patterns, we begin deciphering a genetic network downstream of TGF-β and predict functional links to the control of cell proliferation and EMT

(A) Immunoblot of endogenous SIK and α-tubulin loading control in HaCaT TCL after transfection with control (siLuc) and specific (siSIK) siRNAs and 12 h of stimulation with 5 ng/ml TGFβ1

The asterisk marks a nonspecific protein band. (B) Quantitative real-time RT-PCR analysis in HaCaT cells treated with siRNAs as in A and stimulated with 5 ng/ml TGFβ1 for 6 h ( and ) or 8 h ( and ). Mean fold induction of TGFβ-stimulated mRNA levels (black bars) relative to unstimulated levels (gray bars) are plotted, with standard errors determined from triplicate or quadruplicate samples. Asterisks indicate significant difference ( test: P <p><b>Copyright information:</b></p><p>Taken from "TGFβ induces SIK to negatively regulate type I receptor kinase signaling"</p><p></p><p>The Journal of Cell Biology 2008;182(4):655-662.</p><p>Published online 25 Aug 2008</p><p>PMCID:PMC2518705.</p><p></p

(A) cDNA microarray analysis of mRNA in Smad4-deficient MDA-MB-468 cells after infection with adenovirus expressing LacZ or Smad4 and stimulation with 2 ng/ml TGFβ1 or 300 ng/ml BMP7

Normalized mean expression values with error bars from triplicate microarray expressions are shown (arbitrary units). (B and C) Semiquantitative RT-PCR analysis of and mRNA in MDA-MB-468 cells as in A (B) or in response to 2 ng/ml TGFβ1 in HaCaT cells (C). Amplified cDNA sizes are in bp. (D) SIK and α-tubulin loading control protein profiles in response to 5 ng/ml TGFβ1 in HaCaT cells. Protein sizes are in kilodaltons. (E) Induction of nuclear and cytoplasmic levels of endogenous SIK by 5 ng/ml TGFβ1 in HaCaT cells (bar, 10 μm).<p><b>Copyright information:</b></p><p>Taken from "TGFβ induces SIK to negatively regulate type I receptor kinase signaling"</p><p></p><p>The Journal of Cell Biology 2008;182(4):655-662.</p><p>Published online 25 Aug 2008</p><p>PMCID:PMC2518705.</p><p></p

(A) Coimmunoprecipitation of CA-ALK5 with wild-type (WT) SIK and TCL controls for SIK

Low levels of SIK coexpressed with high levels of CA-ALK5 allowed detection of SIK–ALK5 complexes with only small effects on receptor down-regulation. (B) Coimmunoprecipitation of wild-type SIK with wild type and various Smad7 domains (C, C-terminal; Link, linker; N, N-terminal). TCL controls for SIK and immunoglobulin chains (Ig) are shown. (C) Coimmunoprecipitation of endogenous SIK with endogenous ALK5 and Smad7. HaCaT cell lysates, pretreated with 50 μM MG132 and 5 ng/ml TGFβ1 overnight, were immunoprecipitated with anti-ALK5, anti-Smad7, and anti-SIK (988) antibodies, followed by immunoblotting with anti-SIK (988) antibody. TCL immunoblots with the three antibodies demonstrate specificity of each antiserum. Asterisks indicate specific protein bands and sizes are in kilodaltons. The top left panel (Mock and ALK5) was exposed four times longer than the top right panel (Smad7 and SIK). (D) Triple confocal immunofluorescence analysis of wild-type SIK, CA-ALK5, and wild-type Smad7 in transfected Mv1Lu cells. Insets show high magnifications of peripheral clusters just below the plasma membrane (bar, 10 μm). (E) SIK represses synergistically with Smad7 CAGA promoter induction by 0.5 ng/ml TGFβ1 for 6 h in HepG2 cells transfected with empty vector or Flag-SIK and Flag-Smad7. Normalized promoter activity is plotted as mean values from triplicate determinations with standard error bars. (F) Depletion of endogenous Smad7 stabilizes the CA-ALK5 receptor and wild-type SIK. Immunoblot of HEK293T cell extracts after transient transfection with shRNA vector pSuper-Smad7 or its empty (mock) version together with increasing amounts (triangles) of GFP-SIK and constant amount of CA-ALK5. The asterisks indicate nonspecific protein bands. Protein sizes are shown in kilodaltons.<p><b>Copyright information:</b></p><p>Taken from "TGFβ induces SIK to negatively regulate type I receptor kinase signaling"</p><p></p><p>The Journal of Cell Biology 2008;182(4):655-662.</p><p>Published online 25 Aug 2008</p><p>PMCID:PMC2518705.</p><p></p

The Mechanism of Nuclear Export of Smad3 Involves Exportin 4 and Ran

Transforming growth factor beta (TGF-β) receptors phosphorylate Smad3 and induce its nuclear import so it can regulate gene transcription. Smad3 can return to the cytoplasm to propagate further cycles of signal transduction or to be degraded. We demonstrate that Smad3 is exported by a constitutive mechanism that is insensitive to leptomycin B. The Mad homology 2 (MH2) domain is responsible for Smad3 export, which requires the GTPase Ran. Inactive, GDP-locked RanT24N or nuclear microinjection of Ran GTPase activating protein 1 blocked Smad3 export. Inactivation of the Ran guanine nucleotide exchange factor RCC1 inhibited Smad3 export and led to nuclear accumulation of phosphorylated Smad3. A screen for importin/exportin family members that associate with Smad3 identified exportin 4, which binds a conserved peptide sequence in the MH2 domain of Smad3 in a Ran-dependent manner. Exportin 4 is sufficient for carrying the in vitro nuclear export of Smad3 in cooperation with Ran. Knockdown of endogenous exportin 4 completely abrogates the export of endogenous Smad3. A short peptide representing the minimal interaction domain in Smad3 effectively competes with Smad3 association to exportin 4 and blocks nuclear export of Smad3 in vivo. We thus delineate a novel nuclear export pathway for Smad3