Regulation of TGF-β receptor activity

Background

Thyroid hormone (T3) is important for adult organ function and vertebrate development. Amphibian metamorphosis is totally dependent on T3 and offers a unique opportunity to study how T3 controls postembryonic development in vertebrates. Earlier studies have demonstrated that TR mediates the metamorphic effects of T3 in Xenopus laevis. Liganded TR recruits histone modifying coactivator complexes to target genes during metamorphosis. This leads to nucleosomal removal and histone modifications, including methylation of histone H3 lysine (K) 79, in the promoter regions, and the activation of T3-inducible genes.

Results

We show that Dot1L, the only histone methyltransferase capable of methylating H3K79, is directly regulated by TR via binding to a T3 response element in the promoter region during metamorphosis in Xenopus tropicalis, a highly related species of Xenopus laevis. We further show that Dot1L expression in both the intestine and tail correlates with the transformation of the organs.

Conclusions

Our findings suggest that TR activates Dot1L, which in turn participates in metamorphosis through a positive feedback to enhance H3K79 methylation and gene activation by liganded TR.     

Modification of β-adrenoceptor signal transduction pathway by genetic manipulation and heart failure

The -adrenoceptor (-AR) mediated signal transduction pathway in cardiomyocytes is known to involve ₁- and ₂-ARs, stimulatory (G_s) and inhibitory (G_i) guanine nucleotide binding proteins, adenylyl cyclase (AC) and cAMP-dependent protein kinase (PKA). The activation of ₁- and ₂-ARs has been shown to increase heart function by increasing Ca²⁺-movements across the sarcolemmal membrane and sarcoplasmic reticulum through the stimulation of G_s-proteins, activation of AC and PKA enzymes and phosphorylation of the target sites. The activation of PKA has also been reported to increase phosphorylation of some myofibrillar proteins (for promoting cardiac relaxation) and nuclear proteins (for cardiac hypertrophy). The activation of ₂-AR has also been shown to affect G_i-proteins, stimulate mitogen activated protein kinase and increase protein synthesis by enhancing gene expression. ₁- and ₂-ARs as well as AC are considered to be regulated by PKA- and protein kinase C (PKC)-mediated phosphorylations directly; both PKA and PKC also regulate -AR indirectly through the involvement of -AR kinase (ARK), -arrestins and G-protein subunits. Genetic manipulation of different components and regulators of -AR signal transduction pathway by employing transgenic and knockout mouse models has provided insight into their functional and regulatory characteristics in cardiomyocytes. The genetic studies have also helped in understanding the pathophysiological role of ARK in heart dysfunction and therapeutic role of ARK inhibitors in the treatment of heart failure. Varying degrees of defects in the -AR signal transduction system have been identified in different types of heart failure to explain the attenuated response of the failing heart to sympathetic stimulation or catecholamine infusion. A decrease in ₁-AR density, an increase in the level of G_i-proteins and overexpression of ARK are usually associated with heart failure; however, these attenuations have been shown to be dependent upon the type and stage of heart failure as well as region of the heart. Both local and circulating renin-angiotensin systems, sympathetic nervous system and endothelial cell function appears to regulate the status of -AR signal transduction pathway in the failing heart. Thus different components and regulators of the -AR signal transduction pathway appears to represent important targets for the development of therapeutic interventions for the treatment of heart failure.     

TGF-β singaling and cancer: structural and functional consequences of mutations in Smads

Transforming growth factor-β (TGF-β) and related cytokines control the development and homeostasis of many tissues by regulating the expression of genes that determine cell phenotype. Recent progress has elucidated the way in which members of the TGF-β family initiate their signal through transmembrane receptors and transmit it to target genes via the Smad family of signal-transducing proteins. This review describes TGF-β signaling pathways as currently understood and mutations of the genes that encode Smads that disrupt the function of these proteins and cause various forms of cancer.     

Regulation of autophagy by transforming growth factor-β (TGF-β) signaling

Transforming growth factor-β (TGF-β) has broad impacts on an array of diverse cellular functions including cell growth, differentiation, adhesion, migration, and apoptosis. Perturbations of the TGF-β signaling pathways are involved in progression of various tumors. Autophagy is a pivotal response of normal and cancer cells to environmental stresses and is induced by various stimuli. Otherwise, autophagy has an intrinsic function in tumor suppression. Recently, we demonstrated that TGF-β induces autophagy in hepatocellular carcinoma cells and mammary carcinoma cells. Autophagy activation by TGF-β is mediated through the Smad and JNK pathways. We show that siRNA-mediated knockdown of autophagy genes suppresses the growth inhibitory function of TGF-β and that autophagy activation potentiates TGF-β-mediated induction of proapoptotic genes, Bim and Bmf, in hepatoma cells. In this context, the autophagy pathway might contribute to the growth inhibitory effect of TGF-β, in conjunction with other anti-proliferative pathways downstream of TGF-β signaling. The context and manner by which the TGF-β signaling pathway regulates autophagy have implications for a better understanding of pathological and bidirectional roles of TGF-β signaling pathways in tumorigenesis.     

Regulation of melanin synthesis by the TGF-β family in B16 melanoma cells

Effects of representative members of the transforming growth factor-β (TGF-β) family, TGF-β1, activin A and BMP-2, on melanin content and expression of pigment-producing enzymes were examined in B16 melanoma cells. Treatment with TGF-β1 or activin A but not with BMP-2 significantly decreased melanin content and expression of Tyrosinase and Tyrp-1, suggesting an inhibitory effect of TGF-β1 and activin A on melanin synthesis. TGF-β1 completely inhibited melanin synthesis induced by α-melanin stimulating hormone (α-MSH), whereas activin A only slightly did. As compared with parental B16 cells, the inhibitory effects of TGF-β1 and activin A on melanin content were relative smaller in B16 F10 cells, a subline of B16 cells that contain more pigment. The present study indicates that in addition to TGF-β, activin negatively regulates melanogenesis in the absence of α-MSH, but that the activity in the presence of α-MSH was slightly different between TGF-β and activin.     

Regulation of TCRβ allelic exclusion by gene segment proximity and accessibility

Ag receptor loci are regulated to promote allelic exclusion, but the mechanisms are not well understood. Assembly of a functional TCR β-chain gene triggers feedback inhibition of V(β)-to-DJ(β) recombination in double-positive (DP) thymocytes, which correlates with reduced V(β) chromatin accessibility and a locus conformational change that separates V(β) from DJ(β) gene segments. We previously generated a Tcrb allele that maintained V(β) accessibility but was still subject to feedback inhibition in DP thymocytes. We have now further analyzed the contributions of chromatin accessibility and locus conformation to feedback inhibition using two novel TCR alleles. We show that reduced V(β) accessibility and increased distance between V(β) and DJ(β) gene segments both enforce feedback inhibition in DP thymocytes.     

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.     

Dissecting Arabidopsis Gβ signal transduction on the protein surface

The heterotrimeric G-protein complex provides signal amplification and target specificity. The Arabidopsis (Arabidopsis thaliana) Gβ-subunit of this complex (AGB1) interacts with and modulates the activity of target cytoplasmic proteins. This specificity resides in the structure of the interface between AGB1 and its targets. Important surface residues of AGB1, which were deduced from a comparative evolutionary approach, were mutated to dissect AGB1-dependent physiological functions. Analysis of the capacity of these mutants to complement well-established phenotypes of Gβ-null mutants revealed AGB1 residues critical for specific AGB1-mediated biological processes, including growth architecture, pathogen resistance, stomata-mediated leaf-air gas exchange, and possibly photosynthesis. These findings provide promising new avenues to direct the finely tuned engineering of crop yield and traits.     

Ubiquitin-like protein MNSFβ regulates TLR-2-mediated signal transduction

Post-translational modification by monoclonal nonspecific suppressor factor β (MNSFβ) has been involved in the regulation of a variety of cellular processes. Previous studies have demonstrated that MNSFβ covalently binds to the intracellular pro-apoptotic protein Bcl-G and regulates TLR-4-mediated signal transduction. Recently, we found that MNSFβ also covalently conjugates to endophilin II, a member of the endophilin A family, and inhibits the signal pathway upstream of IKK activation, but not downstream of TLR-2 signaling. In this study, we further examined the mechanism of action of MNSFβ in TLR-2-mediated signal transduction in macrophage-like cell line Raw264.7 cells. Although MNSFβ siRNA enhanced Pam(3)CDK(4) (TLR-2-specific ligand)-stimulated TNFα production, Bcl-G siRNA did not affect. MNSFβ cDNA inhibited the Pam(3)CDK(4)-stimulated TNFα production. High-molecular weight (130 kDa) MNSFβ-adduct was induced in Pam(3)CDK(4)-stimulated Raw264.7 cells. This MNSFβ-adduct was not induced by LPS, indicative of the specificity of TLR-2-mediated signal transduction. Similar observations were seen in BALB/c peritoneal macrophages. Interestingly, 40-kDa MNSFβ-adduct was tyrosine phosphorylated by Pam(3)CDK(4) stimulation. Collectively, novel MNSFβ-adducts may regulate TLR-2 signaling pathway in macrophages.     

Regulation of amyloid-β production by the prion protein

Alzheimer disease (AD) is characterized by the amyloidogenic processing of the amyloid precursor protein (APP), culminating in the accumulation of amyloid-β peptides in the brain. The enzymatic action of the β-secretase, BACE1 is the rate-limiting step in this amyloidogenic processing of APP. BACE1 cleavage of wild-type APP (APP_WT) is inhibited by the cellular prion protein (PrP^C). Our recent study has revealed the molecular and cellular mechanisms behind this observation by showing that PrP^C directly interacts with the pro-domain of BACE1 in the trans-Golgi network (TGN), decreasing the amount of BACE1 at the cell surface and in endosomes where it cleaves APP_WT, while increasing BACE1 in the TGN where it preferentially cleaves APP with the Swedish mutation (APP_Swe). PrP^C deletion in transgenic mice expressing the Swedish and Indiana familial mutations (APP_Swe,Ind) failed to affect amyloid-β accumulation, which is explained by the differential subcellular sites of action of BACE1 toward APP_WT and APP_Swe. This, together with our observation that PrP^C is reduced in sporadic but not familial AD brain, suggests that PrP^C plays a key protective role against sporadic AD. It also highlights the need for an APP_WT transgenic mouse model to understand the molecular and cellular mechanisms underlying sporadic AD.     

Regulation of TGFβ receptor trafficking and signaling by atypical protein kinase C

Transforming growth factor beta (TGFβ) signaling is linked to the membrane trafficking of TGFβ receptors. The Protein Kinase C (PKC) family of serine/threonine kinases have been implicated in modulating the endocytic processes of various receptors. The present study investigated whether PKC activity plays a role in the trafficking, and signaling of TGFβ receptors, and further explored which PKC isoforms may be responsible for altered TGFβ signaling patterns. Using immunofluorescence microscopy and ¹²⁵I-TGFβ internalization assays, we show that the pharmacological inhibition of PKC activity alters TGFβ receptor trafficking and delays TGFβ receptor degradation. Consistent with these findings, we demonstrate that PKC inhibition extends TGFβ-dependent Smad2 phosphorylation. Previous studies have shown that PKCζ associates with TGFβ receptors to modulate cell plasticity. We therefore used siRNA directed at the atypical PKC isoforms to investigate if reducing PKCι and PKCζ protein levels would delay TGFβ receptor degradation and extend TGFβ signaling. Our findings suggest that atypical PKC isoforms regulate TGFβ signaling by altering cell surface TGFβ receptor trafficking and degradation.     

TMEPAI inhibits TGF-β signaling by promoting lysosome degradation of TGF-β receptor and contributes to lung cancer development

Transforming growth factor-β (TGF-β) signaling plays important roles in embryogenesis and tumorigenesis by controlling cell growth, differentiation and migration. The transmembrane prostate androgen-induced protein (TMEPAI) is elevated in several cancers. TMEPAI expression is induced by TGF-β signaling, and in turn, expression of TMEPAI negatively regulates TGF-β signaling, but the molecular mechanisms of TMEPAI induced TGF-β signaling inhibition are not well understood. Here we report that TMEPAI is localized to the lysosome and late endosome, and that association of TMEPAI with the E3 ubiquitin ligase Nedd4 is required for its transport to the lysosome. TMEPAI associates with the TGF-β type I receptor (TβRI) and promotes its degradation in the lysosome. Depletion of TMEPAI in A549 lung cancer cells inhibits cell proliferation, migration and invasion, while TMEPAI expression in nude mice promotes tumorigenesis. These results reveal a novel function for TMEPAI in regulating TGF-β signaling through the modulation of TβRI levels, which has important implications for cancer development in vivo.     

TGF-β sensitivity is determined by N-linked glycosylation of the type II TGF-β receptor

N-linked glycosylation is a critical determinant of protein structure and function, regulating processes such as protein folding, stability and localization, ligand-receptor binding and intracellular signalling. TβRII [type II TGF-β (transforming growth factor β) receptor] plays a crucial role in the TGF-β signalling pathway. Although N-linked glycosylation of TβRII was first demonstrated over a decade ago, it was unclear how this modification influenced TβRII biology. In the present study, we show that inhibiting the N-linked glycosylation process successfully hinders binding of TGF-β1 to TβRII and subsequently renders cells resistant to TGF-β signalling. The lung cancer cell line A549, the gastric carcinoma cell line MKN1 and the immortal cell line HEK (human embryonic kidney)-293 exhibit reduced TGF-β signalling when either treated with two inhibitors, including tunicamycin (a potent N-linked glycosylation inhibitor) and kifunensine [an inhibitor of ER (endoplasmic reticulum) and Golgi mannosidase I family members], or introduced with a non-glycosylated mutant version of TβRII. We demonstrate that defective N-linked glycosylation prevents TβRII proteins from being transported to the cell surface. Moreover, we clearly show that not only the complex type, but also a high-mannose type, of TβRII can be localized on the cell surface. Collectively, these findings demonstrate that N-linked glycosylation is essentially required for the successful cell surface transportation of TβRII, suggesting a novel mechanism by which the TGF-β sensitivity can be regulated by N-linked glycosylation levels of TβRII.