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PubMed2002

Beta-catenin, MAPK and Smad signaling during early Xenopus development.

Knowledge of when and where signaling pathways are activated is crucial for understanding embryonic development. In this study, we have systematically analyzed and compared the signaling pattern of four major pathways by localization of the activated key components beta-catenin (Wnt proteins), MAPK (tyrosine kinase receptors/FGF), Smad1 (BMP proteins) and Smad2 (Nodal/activin/Vg1). We have determined semi-quantitatively the distribution of these components at 18 consecutive stages in Xenopus development, from early blastula to tailbud stages, by immunofluorescence on serial cryosections. The image obtained is that of very dynamic and widespread activities, with very few inactive regions. Signaling fields can vary from large gradients to restricted areas with sharp borders. They do not respect tissue boundaries. This direct visualization of active signaling verifies several predictions inferred from previous functional data. It also reveals unexpected signal patterns, pointing to some poorly understood aspects of early development. In several instances, the patterns strikingly overlap, suggesting extensive interplay between the various pathways. To test this possibility, we have manipulated maternal beta-catenin signaling and determined the effect on the other pathways in the blastula embryo. We found that the patterns of P-MAPK, P-Smad1 and P-Smad2 are indeed strongly dependent on beta-catenin at this stage.

Animals↗
PubMedDate not supplied

Expression and intracellular localization of Smad proteins in human endometrial cancer.

The aim of our study was to examine expression of Smad proteins i.e., Smad2, Smad3 and Smad4 both as mRNA and protein as well as their intracellular localization in normal (n=13) and neoplastic (n=42) endometrial tissue specimens using RT-PCR and immunological techniques i.e., Western blot and ELISA. Two uncommon female genital tract tumours, rhabdomyosarcoma of uterine of the cervix and uterine carcinosarcoma were also included. No statistically significant differences were found in the mRNA level of the examined Smad proteins between normal and tumour tissue specimens. Smad2 and Smad3 mRNAs were detected both in uterine carcinosarcoma and rhabdomyosarcoma of the uterine cervix. However, significantly lower Smad2 and Smad4 mRNA level was noted when the depth of myometrial invasion was considered (p<0.05). In endometrial cancer as compared to normal endometrium significantly higher levels of Smad2 and Smad3 proteins, both in cytoplasmic (p=0.002; p=0.0001) and nuclear (p=0.016; p=0.0004) fractions were observed. Both in uterine carcinosarcoma and rhabdomyosarcoma of the uterine cervix Smad2, Smad3 and Smad4 proteins were not detected. Moreover, significantly elevated Smad4 protein level in cytoplasmic fraction was stated when tumour grade and depth of myometrial invasion was undertaken (p<0.05). When intracellular distribution of Smads was considered differences between cytoplasmic and nuclear localization in normal and carcinomatous endometrium was stated. In endometrial cancer decreased number of cases with Smad3 and increased number of cases with Smad4 located in nuclear fraction was found. In conclusion, the disturbances in Smad protein expression and/or differences in their intracellular distribution suggest, that TGF-beta signaling pathway via Smads may be deregulated in endometrial carcinomas.

Blotting, Western↗
PubMed1999

Mad2 binding by phosphorylated kinetochores links error detection and checkpoint action in mitosis.

The spindle checkpoint must detect the presence of unattached or improperly attached kinetochores and must then inhibit progression through the cell cycle until the offending condition is resolved. Detection probably involves attachment-sensitive kinetochore phosphorylation (reviewed in [1,2]). A key player in the checkpoint's response is the Mad2 protein, which prevents activation of the anaphase-promoting complex (APC) by the Cdc20 protein [3-8]. Microinjection of Mad2 antibodies results in premature anaphase onset [9,10], and excess Mad2 protein causes arrest in mitosis [5,11]. We have previously shown that Mad2 localizes to unattached kinetochores in vertebrate cells, and that this localization ceases as kinetochores accumulate microtubules [10,12,13]. But how is Mad2 binding limited to unattached kinetochores? Here, we used lysed PtK1 cells to study kinetochore phosphorylation and Mad2 binding. We found that Mad2 binds to phosphorylated kinetochores, but not to unphosphorylated ones. Our data suggest that it is kinetochore protein phosphorylation that promotes Mad2 binding to unattached kinetochores. Thus, we have identified a probable molecular link between attachment-sensitive kinetochore phosphorylation and the inhibition of anaphase. The complete pathway for error control in mitosis can now be outlined.

Animals↗
PubMed2003

The comparative role of activator protein 1 and Smad factors in the regulation of Timp-1 and MMP-1 gene expression by transforming growth factor-beta 1.

The balance between matrix metalloproteinases (MMPs) and their inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), is pivotal in the remodeling of extracellular matrix. TGF-beta has profound effects on extracellular matrix homeostasis, in part via its ability to alter this balance at the level of gene expression. The intracellular signaling pathways by which TGF-beta mediates its actions include the Smad pathway, specific to the TGF-beta superfamily, but also, for example, mitogen-activated protein kinase pathways; furthermore, cross-talk between the Smads and other signaling pathways modifies the TGF-beta response. The reciprocal effect of TGF-beta on the expression of Timp-1 and MMP-1 supports its role in matrix anabolism, yet the mechanisms by which TGF-beta induces Timp-1 and represses induced MMP-1 have remained opaque. Here, we (i) investigate the mechanism(s) by which TGF-beta1 induces expression of the Timp-1 gene and (ii) compare this with TGF-beta1 repression of phorbol ester-induced MMP-1 expression. We report that the promoter-proximal activator protein 1 (AP1) site is essential for the response of both Timp-1 and MMP-1 to TGF-beta (induction and repression, respectively). c-Fos, JunD, and c-Jun are essential for the induction of Timp-1 gene expression by TGF-beta1, but these AP1 factors transactivate equally well from both Timp-1 and MMP-1 AP1 sites. Smad-containing complexes do not interact with the Timp-1 AP1 site, and overexpression of Smads does not substitute or potentiate the induction of the gene by TGF-beta1; furthermore, Timp-1 is still induced by TGF-beta1 in Smad knockout cell lines, although to varying extents. In contrast, Smads do interact with the MMP-1 AP1 site and mediate repression of induced MMP-1 gene expression by TGF-beta1.

Animals↗
PubMed2005

Influence of exogenous TGFbeta1 on the expression of smad2 and smad3 in rat bone marrow-derived mesenchymal stem cells.

The expression of Smad2 and Smad3 and the influence of exogenous transforming growth factorbeta1 (TGFbeta1) on them in rat bone marrow-derived mesenchymal stem cells (MSCs) cultured in vitro were investigated. The effects of different concentrations of TGFbeta1 on cell proliferation and ALP activity were detected by MTT and PNPP in MSCs respectively. The expression of Smad2 and Smad3 and the influence of exogenous TGFbeta1, on them were also examined by immunocytochemistry and Western blot assays. The exogenous TGFbeta1 induced a dose-dependent decrease in cell proliferation and a dose-dependent increase in ALP activity, which plateaued at 5 ng/ml. Smad2 and Smad3 proteins were detected only in the cytoplasm in the absence of TGFbeta1, and TGFbeta1, could stimulate the translocation of them from the cytoplasm to the nucleus. The total amount of Smad2 protein remained unchanged before and after TGFbeta1 treatment (P > 0.05). The expression levels of Smad3 remained unchanged after 3 h and 6 h treatment (P > 0.05), but decreased markedly after 24 h treatment (P <0.05). It was concluded that TGFbeta1 is a latent osteoinductive factor involved in osteoblastic differentiation. Both Samd2 and Smad3 mediate TGFbeta1 signaling as downstream mediators in MSCs. The biological output of TGFbeta1 triggering the osteoblastic differentiation could be entirely determined by Smad3 in MSCs.

Animals↗
PubMed2000

Molecular mechanisms of osteolytic bone metastases.

BACKGROUND: Breast carcinoma commonly metastasizes to the skeleton in patients with advanced disease to cause bone destruction and the associated pain, hypercalcemia, fracture, and nerve-compression syndromes. In this scenario, the bone destruction is mediated by the osteoclast. Tumor-produced parathyroid hormone-related protein (PTHrP), a known stimulator of osteoclastic bone resorption, is a major mediator of the osteolytic process. Transforming growth factor beta (TGFbeta), which is abundant in bone matrix and is released as a consequence of osteoclastic bone resorption, may promote breast carcinoma osteolysis by stimulating PTHrP production by tumor cells. METHODS: Stable breast carcinoma MDA-MB-231 cell lines were constructed that expressed mutant TGFbeta receptors, Smad proteins, or estrogen receptor (ER)-alpha and were used to determine the role of TGFbeta in modulating tumor production of PTHrP. These stable cell lines were applied to a mouse model of human breast carcinoma metastases to the bone to dissect the molecular mechanisms responsible for osteolytic bone metastases. RESULTS: TGFbeta promoted the development and progression of osteolytic bone metastases by inducing tumor production of PTHrP, the effect of which was mediated through the Smad signaling pathway. PTHrP stimulated osteoclastic bone resorption by increasing osteoblast production of the receptor activator of nuclear factor K B (RANK) ligand and decreasing osteoblast production of osteoprotegerin (OPG). A constitutively active ER-alpha mutation (Tyr537Asn), identified from a human bone metastases, when it was expressed in human breast carcinoma cells, caused increased production of PTHrP. TGFbeta significantly enhanced the ER-alpha-mediated transcriptional activity induced by ER-alpha (Tyr537Asn), and this resulted in further stimulation of PTHrP production. CONCLUSIONS: These data indicate a central role for TGFbeta in the pathogenesis of osteolytic bone metastases from breast carcinoma by 1) the induction of PTHrP through the Smad signaling pathway and 2) the potentiation of ER-alpha-mediated transcription induced by a constitutively active ER-alpha. Understanding the mechanisms of osteolysis at a molecular level will generate more effective therapeutic agents for patients with this devastating complication of cancer.

Animals↗
PubMed2003

Repression of Smad-dependent transforming growth factor-beta signaling by Epstein-Barr virus latent membrane protein 1 through nuclear factor-kappaB.

EBV-encoded LMP-1 is absolutely required for EBV transformation of cells. Previous studies showed that LMP-1 is responsible for mediating resistance to the anti-proliferative effects of TGF-beta that characterizes EBV-transformed cells. To clarify the mechanisms of resistance to TGF-beta by LMP-1, we examined the effect of expression of LMP-1 on the activity of TGF-beta-responsive promoters. Interestingly, LMP-1 inhibited TGF-beta-responsive promoters activity despite lack of direct interaction of LMP-1 and Smad proteins, intracellular signaling molecules in the TGF-beta signal transduction pathway. Although TGF-beta treatment increased the expression of p15, TGF-beta-induced gene, this effect was counteracted by expression of LMP-1. The repressive effect was mapped to the NF-kappaB activation domains in the cytoplasmic carboxyl terminus of LMP-1. Furthermore, LMP-1-mediated inhibition of TGF-beta-responsive promoter was markedly restored after inhibition of NF-kappaB activity. LMP-1 failed to affect receptor-dependent formation of heteromers containing Smad proteins as well as the DNA-binding activity of Smad proteins. Overexpression of the transcriptional coactivator CBP and p300 abrogated the inhibitory effect of LMP-1 on the TGF-beta-responsive promoter. Our results suggest that LMP-1 represses the TGF-beta signaling through the NF-kappaB signaling pathway at transcriptional level by competing for a limited pool of transcriptional coactivators. These results enhance our understanding of the molecular mechanisms of viral pathogenesis in EBV-associated malignancies.

Animals↗
PubMed2004

SPARC regulates TGF-beta1-dependent signaling in primary glomerular mesangial cells.

Secreted protein acidic and rich in cysteine (SPARC), a member of the family of matricellular proteins, regulates the interaction of cells with pleiotropic factors and proteins of the extracellular matrix (ECM). Although it has been appreciated that transforming growth factor beta 1 (TGF-beta1) induces SPARC and collagen type I, we have recently shown that SPARC regulates the expression of TGF-beta1 and collagen type I in renal mesangial cells via a TGF-beta1-dependent pathway, and have proposed a reciprocal, autocrine regulatory feedback loop between SPARC and TGF-beta1. Herein, we sought to determine how SPARC regulates TGF-beta1-dependent signal transduction. Our data indicate that SPARC modulates the TGF-beta1-dependent phosphorylation of Smad-2 in primary mesangial cells derived from wild-type and SPARC-null mice. We also show that SPARC regulates the levels and activation of the stress-activated c-jun-N-terminal kinase (JNK) in mesangial cells by augmentation of the stimulatory effects of TGF-beta1. Furthermore, we found that SPARC increases the levels and the activity of the transcription factor c-jun. These effects of SPARC on the TGF-beta1 signaling pathway appear to be mediated through an interaction with the TGF-beta1-receptor complex, but only in the presence of TGF-beta1 bound to its cognate type II receptor. That SPARC is directly involved in the regulation of the TGF-beta1 signaling cascade is consistent with the paradigm that matricellular proteins modulate interactions among cells, growth factors, and their respective receptors.

Animals↗
PubMed2006

Reduction of invasive potential in K-ras-transformed thyroid cells by restoring of TGF-beta pathway.

Transforming Growth Factor-beta1 (TGF -beta1) is a multifunctional cytokine that regulates a number of cellular processes such as cell growth, differentiation, plasticity, cell motility, adhesiveness, embryogenesis, development and apoptosis through binding to TGF-beta receptors. We have previously demonstrated that K-ras-transformed rat thyroid cells, K10, are resistant to the growth inhibitory action of TGF-beta1, because they show a decreased expression of type II receptor (TbetaRII). Clones obtained transfecting TbetaRII, partially revert their malignant phenotype, showing a reduction in the anchorage-dependent and -independent cell growth and a statistically significant decrease in tumourigenicity with respect to the highly malignant parental cells, both in spontaneous and artificial metastases, when transplanted in athymic nude mice. The purpose of the present work is to elucidate the molecular events involved in the modulation of the tumourigenic potential of K-ras-transformed rat thyroid cells overexpressing TbetaRII. Our data demonstrate that the TbetaRII overexpressed in K-ras-transformed thyroid cell clones is a functional receptor and is essential to restore in these cells behaviour similar to that of control cells. The TbetaRII overexpression is responsible for a strong reduction of adhesive and migratory behaviour of highly malignant K-ras-transformed thyroid cells. These results suggest that the restore of a functional TGF-beta receptor in these cells may be useful for the limitation of tumour spread and dissemination.

Animals↗
PubMed2006

Induction of TIMP-1 and HSP47 synthesis in primary keloid fibroblasts by exogenous nitric oxide.

BACKGROUND: The excessive accumulation of extracellular matrix is a hallmark of many fibrotic diseases, including the hypertrophic scar and keloid. Recent reports from this research team had shown that exogenous nitric oxide (NO) participates in the keloid formation; however, its role on the synthesis of fibrotic factor (TGF-beta1, TIMP-1 and HSP47) in the keloid fibroblasts (KF) remained unclear. OBJECTIVE: In this study, to better define the potential effect of exogenous NO on the expression of fibrotic factors in KF, the enhancing effect of exogenous NO, released from a NO donor, on the synthesis of fibrotic factors in KF was investigated. METHODS: The seven primary KF cultures were set up to measure the effect of exogenous NO on enhancing the expression of fibrotic factor. RESULTS: Elevation of cellular cGMP levels was observed to be induced by NO or blocked by the hydrolysis activity of phosphodiesterase (PDE) by the PDE inhibitor. The elevated levels of cellular cGMP were noted to enhance the expression of TIMP-1 and HSP47 in KF. Exogenous NO was found to significantly accelerate the production of TIMP-1 and HSP47 in the seven primary KFs with a corresponding increase in the production of TGF-beta1. CONCLUSION: The results have led to a conclusion, that is: the excess collagen formations in the keloid lesion may be attributed to the NO/cGMP signal pathway by initiating a rapid increase in the expression of TGF-beta1, TIMP-1 and HSP47 in the KF cells.

Adult↗
PubMed2000

Signaling inputs converge on nuclear effectors in TGF-beta signaling.

Recent studies have consolidated the pivotal role of Smads as intracellular effectors of TGF-beta family members. Upon binding to their specific type I and type II serine/threonine kinase receptors, each family member activates a particular subset of Smad proteins. Activated, receptor-regulated Smads form hetero-oligomeric complexes with common-partner Smads that translocate into the nucleus, where they control the expression of target genes in a cell-type-specific manner. Smads appear to function not only as nuclear effectors for TGF-beta family members, but as signal integrators within an extensive intracellular network.

Animals↗
PubMed2004

Cyclin-dependent kinases regulate the antiproliferative function of Smads.

Transforming growth factor-beta (TGF-beta) potently inhibits cell cycle progression at the G1 phase. Smad3 has a key function in mediating the TGF-beta growth-inhibitory response. Here we show that Smad3 is a major physiological substrate of the G1 cyclin-dependent kinases CDK4 and CDK2. Except for the retinoblastoma protein family, Smad3 is the only CDK4 substrate demonstrated so far. We have mapped CDK4 and CDK2 phosphorylation sites to Thr 8, Thr 178 and Ser 212 in Smad3. Mutation of the CDK phosphorylation sites increases Smad3 transcriptional activity, leading to higher expression of the CDK inhibitor p15. Mutation of the CDK phosphorylation sites of Smad3 also increases its ability to downregulate the expression of c-myc. Using Smad3(-/-) mouse embryonic fibroblasts and other epithelial cell lines, we further show that Smad3 inhibits cell cycle progression from G1 to S phase and that mutation of the CDK phosphorylation sites in Smad3 increases this ability. Taken together, these findings indicate that CDK phosphorylation of Smad3 inhibits its transcriptional activity and antiproliferative function. Because cancer cells often contain high levels of CDK activity, diminishing Smad3 activity by CDK phosphorylation may contribute to tumorigenesis and TGF-beta resistance in cancers.

Animals↗
PubMed2004

TGF-beta and HGF transmit the signals through JNK-dependent Smad2/3 phosphorylation at the linker regions.

Although hepatocyte growth factor (HGF) can act synergistically or antagonistically with transforming growth factor-beta (TGF-beta) signaling, molecular mechanism of their crosstalk remains unknown. Using antibodies which selectively distinguished receptor-regulated Smads (R-Smads) phosphorylated at linker regions from those at C-terminal regions, we herein showed that either HGF or TGF-beta treatment of normal stomach-origin cells activated the JNK pathway, thereafter inducing endogenous R-Smads phosphorylation at linker regions. However, the phosphorylation at their C-terminal regions was not induced by HGF treatment. The activated JNK could directly phosphorylate R-Smads in vitro at the same sites that were phosphorylated in response to TGF-beta or HGF in vivo. Thus, the linker regions of R-Smads were the common phosphorylation sites for HGF and TGF-beta signaling pathways. The phosphorylation induced by simultaneous treatment with HGF and TGF-beta allowed R-Smads to associate with Smad4 and to translocate into the nucleus. JNK pathway involved HGF and TGF-beta-mediated infiltration potency since a JNK inhibitor SP600125 caused the reduction of invasive capacity induced by HGF and TGF-beta signals. Moreover, a combined treatment with HGF and TGF-beta led to a potent increase in plasminogen activator inhibitor type 1 transcriptional activity through Smad3 phosphorylation at the linker region. In contrast, HGF treatment reduced TGF-beta-dependent activation of p15INK4B promoter, in which Smad3 phosphorylation at the C-terminal region was involved. In conclusion, HGF and TGF-beta transmit the signals through JNK-mediated R-Smads phosphorylation at linker regions.

Animals↗
PubMed2004

The Kaposi's sarcoma-associated herpesvirus K-bZIP protein represses transforming growth factor beta signaling through interaction with CREB-binding protein.

Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) is involved in the pathogenesis of KS, primary effusion lymphoma, and multicentric Castleman's disease. K-bZIP, the protein encoded by the open reading frame K8 of KSHV, is a member of the basic region-leucine zipper family of transcription factors. We studied the mechanisms that underlie KSHV-induced oncogenesis by investigating whether K-bZIP perturbs signaling through transforming growth factor beta (TGF-beta), which inhibits proliferation of a wide range of cell types. K-bZIP repressed TGF-beta-induced, Smad-mediated transcriptional activity and antagonized the growth-inhibitory effects of TGF-beta. Since both K-bZIP and Smad are known to interact with CREB-binding protein (CBP), the effect of CBP on inhibition of Smad-mediated transcriptional activation by K-bZIP was examined. K-bZIP mutants, which lacked the CBP-binding site, could not repress TGF-beta-induced or Smad3-mediated transcriptional activity. Overexpression of CBP restored K-bZIP-induced inhibition of Smad3-mediated transcriptional activity. Competitive interaction studies showed that K-bZIP inhibited the interaction of Smad3 with CBP. These results suggest that K-bZIP, through its binding to CBP, disrupts TGF-beta signaling by interfering with the recruitment of CBP into transcription initiation complexes on TGF-beta-responsive elements. We propose a possibility that K-bZIP may contribute to oncogenesis through its ability to promote cell survival by repressing TGF-beta signaling.

Base Sequence↗
PubMed1998

Smad proteins physically interact with calmodulin.

The Smad family of intracellular proteins mediates signals generated by activin and other transforming growth factor beta-related proteins via specific heteromeric complexes of transmembrane receptor serine kinases (1, 2). xSmad2 has been implicated as an activin signal mediator that may participate in transcriptional regulation (3, 4). We have employed an interaction cloning strategy to identify xSmad2-binding proteins and found that calmodulin directly associated with Smads. xSmad2, generated either by in vitro translation or by overexpression in COS cells, specifically bound to calmodulin-agarose; the association was calcium-dependent and required xSmad2 N-terminal residues. In the same assay, xSmad1 and hSmads 2, 3, and 4 also bound to calmodulin-agarose. Furthermore, a calmodulin antagonist, W13, increased expression of the activin-inducible transcriptional reporter, 3TP-Lux, whereas overexpression of calmodulin suppressed this reporter. These observations demonstrate that Smad proteins interact with calmodulin in a calcium-dependent way through conserved N-terminal amino acids and suggest a role for calmodulin in regulating Smad function.

Activins↗
PubMed2000

The Smad4 activation domain (SAD) is a proline-rich, p300-dependent transcriptional activation domain.

Transforming growth factor-beta (TGF-beta) family members signal through a unique set of intracellular proteins called Smads. Smad4, previously identified as the tumor suppressor DPC4, is functionally distinct among the Smad family, and is required for the assembly and transcriptional activation of diverse, Smad-DNA complexes. We previously identified a 48-amino acid proline-rich regulatory element within the middle linker domain of this molecule, the Smad4 activation domain (SAD), which is essential for mediating these signaling activities. We now characterize the functional activity of the SAD. Mutants lacking the SAD are still able to form complexes with other Smad family members and associated transcription factors, but cannot activate transcription in these complexes. Furthermore, the SAD itself is able to activate transcription in heterologous reporter assays, identifying it as a proline-rich transcriptional activation domain, and indicating that the SAD is both necessary and sufficient to activate Smad-dependent transcriptional responses. We show that transcriptional activation by the SAD is p300-dependent, and demonstrate that this activity is associated with a physical interaction of the SAD with the amino terminus of p300. These data identify a novel function of the middle linker region of Smad4, and define the role of the SAD as an important locus determining the transcriptional activation of the Smad complex.

Amino Acid Sequence↗