Cloning and expression of a rat Smad1: Regulation by TGFß and modulation by the ras/MEK pathway

A new family of signaling intermediates for TGFß superfamily members and other growth factors has recently been identified and termed Smads. It has been suggested that the Smad1 subfamily is regulated primarily by the TGFß superfamily member bone morphogenetic protein (BMP). Here we demonstrate that TGFß induced phosphorylation of endogenous Smad1 in untransformed IECs and that the RI and RII TGFß receptors were detectable in Smad1 immunocomplexes. Expression of a dominant-negative mutant of Ras inhibited the ability of TGFß to phosphorylate endogenous Smad1. In a separate series of experiments, we have cloned a rat homologue of the drosophila mad gene (termed RSmad1) by screening an intestinal epithelial cell (IEC) cDNA library. By using an in vitro kinase assay with RSmad1 as the substrate, we demonstrate that the TGFß receptor complex can directly phosphorylate RSmad1. We show, further, that a dominant-negative mutant of MEK1 inhibited the ability of RSmad1 to induce the TGFß-responsive reporter p3TP-Lux in a human breast cancer cell line. Collectively, our data demonstrate that TGFß can regulate Smad1 and that the Ras and MEK signaling components are partially required for the ability of TGFß to regulate Smad1. J. Cell. Physiol. 178:387–396, 1999. © 1999 Wiley-Liss, Inc.     

Restoration of interleukin-2 production in tumor-bearing rats through reducing tumor-derived transforming growth factor β by treatment with bleomycin

We studied mechanisms of immunosuppression caused by tumor-derived transforming growth factor-ß (TGFß) and restoration of the immune response by treatment with bleomycin in rats bearing KDH-8 hepatoma. Interleukin-2 (IL-2) production from splenocytes of KDH-8-tumor-bearing rats progressively decreased as the KDH-8 tumor grew. IL-2 production from concanavalin-A-stimulated normal rat splenocytes was signficiantly inhibited by in vitro cultured KDH-8-tumor-cell-conditioned medium; this inhibition could be blocked by neutralizing the conditioned medium with anti-TGFß antibody. TGFß activities were found in KDH-8-tumor-tissue-conditioned medium without acid treatment and were found in tumor-cell-conditioned medium after acid treatment; TGFß mRNA and TGFß protein were found in cultured KDH-8 tumor cells. These results suggested that the KDH-8-tumor-derived TGFß might be involved in the inhibition of IL-2 production from splenocytes. To determine whether bleomycin chemotherapy could reduce tumor-derived TGFß and restore the immune responses, we treated KDH-8 tumor-bearing rats with bleomycin (5 mg/kg, one shot) at an appropriate time (before the occurrence of immunosuppression) resulting in a significiant reduction of TGFß activity in KDH-8 tumor tissues and restoration of IL-2 production from splenocytes of tumor-bearing rats; KDH-8 tumor growth ultimately regressed. In vitro experiments also showed that TGFß activity, mRNA expression, and protein synthesis in KDH-8 tumor cells were reduced by bleomycin treatment, and that bleomycin-treated-KDH-8-tumor-cell-conditioned medium did not inhibit IL-2 production from normal rat splenocytes. These results suggest that bleomycin treatment restored IL-2 production in tumor-bearing rats through reducing the tumor-derived TGFß.     

The BRAFV600E inhibitor,PLX4032, increases type I collagen synthesis in melanoma cells

Vertical growth phase (VGP) melanoma is frequently metastatic, a process mediated by changes in gene expression, which are directed by signal transduction pathways in the tumor cells. A prominent signaling pathway is the Ras-Raf-Mek-Erk MAPK pathway, which increases expression of genes that promote melanoma progression. Many melanomas harbor a mutation in this pathway, BRAF^V600E, which constitutively activates MAPK signaling and expression of downstream target genes that facilitate tumor progression. In BRAF^V600E melanoma, the small molecule inhibitor, vemurafenib (PLX4032), has revolutionized therapy for melanoma by inducing rapid tumor regression. This compound down-regulates the expression of many genes. However, in this study, we document that blocking the Ras-Raf-Mek-Erk MAPK pathway, either with an ERK (PLX4032) or a MEK (U1026) signaling inhibitor, in BRAF^V600E human and murine melanoma cell lines increases collagen synthesis in vitro and collagen deposition in vivo. Since TGFß signaling is a major mediator of collagen synthesis, we examined whether blocking TGFß signaling with a small molecule inhibitor would block this increase in collagen. However, there was minimal reduction in collagen synthesis in response to blocking TGFß signaling, suggesting additional mechanism(s), which may include activation of the p38 MAPK pathway. Presently, it is unclear whether this increased collagen synthesis and deposition in melanomas represent a therapeutic benefit or an unwanted “off target” effect of inhibiting the Ras-Raf-Erk-Mek pathway.     

Arylstibonic acids are potent and isoform-selective inhibitors of Cdc25a and Cdc25b phosphatases

Arylstibonates structurally resemble phosphotyrosine side chains in proteins and here we addressed the ability of such compounds to act as inhibitors of a panel of mammalian tyrosine and dual-specificity phosphatases. Two arylstibonates both possessing a carboxylate side chain were identified as potent inhibitors of the protein tyrosine phosphatase PTP-ß. In addition, they inhibited the dual-specificity, cell cycle regulatory phosphatases Cdc25a and Cdc25b with sub-micromolar potency. However, the Cdc25c phosphatase was not affected demonstrating that arylstibonates may be viable leads from which to develop isoform specific Cdc25 inhibitors.     

Human papillomavirus type 5 E6 oncoprotein represses the transforming growth factor beta signaling pathway by binding to SMAD3

Mechanisms of cellular transformation associated with human papillomavirus type 5 (HPV5), which is responsible for skin carcinomas in epidermodysplasia verruciformis (EV) patients, are poorly understood. Using a yeast two-hybrid screening and molecular and cellular biology experiments, we found that HPV5 oncoprotein E6 interacts with SMAD3, a key component in the transforming growth factor beta1 (TGF-beta1) signaling pathway. HPV5 E6 inhibits SMAD3 transactivation by destabilizing the SMAD3/SMAD4 complex and inducing the degradation of both proteins. Interestingly, the E6 protein of nononcogenic EV HPV9 failed to interact with SMAD3, suggesting that downregulation of the TGF-beta1 signaling pathway could be a determinant in HPV5 skin carcinogenesis.     

Dual requirement of ectodermal Smad4 during AER formation and termination of feedback signaling in mouse limb buds

BMP signaling is pivotal for normal limb bud development in vertebrate embryos and genetic analysis of receptors and ligands in the mouse revealed their requirement in both mesenchymal and ectodermal limb bud compartments. In this study, we genetically assessed the potential essential functions of SMAD4, a mediator of canonical BMP/TGFß signal transduction, in the mouse limb bud ectoderm. Msx2‐Cre was used to conditionally inactivate Smad4 in the ectoderm of fore‐ and hindlimb buds. In hindlimb buds, the Smad4 inactivation disrupts the establishment and signaling by the apical ectodermal ridge (AER) from early limb bud stages onwards, which results in severe hypoplasia and/or aplasia of zeugo‐ and autopodal skeletal elements. In contrast, the developmentally later inactivation of Smad4 in forelimb buds does not alter AER formation and signaling, but prolongs epithelial‐mesenchymal feedback signaling in advanced limb buds. The late termination of SHH and AER‐FGF signaling delays distal progression of digit ray formation and inhibits interdigit apoptosis. In summary, our genetic analysis reveals the temporally and functionally distinct dual requirement of ectodermal Smad4 during initiation and termination of AER signaling. genesis 51:660–666. © 2013 Wiley Periodicals, Inc.     

Distinct oligomeric states of SMAD proteins in the transforming growth factor-beta pathway

Protein interactions are critical for the function of SMADs as mediators of transforming growth factor-beta (TGF-beta) signals. TGF-beta receptor phosphorylation of SMAD2 or SMAD3 causes their association with SMAD4 and accumulation in the nucleus where the SMAD complex binds cofactors that determine the choice of target genes. We provide evidence that in the basal state, SMADs 2, 3, and 4 form separate, strikingly different complexes. SMAD2 is found mostly as monomer, whereas the closely related SMAD3 exists in multiple oligomeric states. This difference is due to a unique structural element in the MH1 domain of SMAD2 that inhibits protein-protein interactions in the basal state. In contrast to SMAD2 and SMAD3, SMAD4 in the basal state is found mostly as a homo-oligomer, most likely a trimer. Upon cell stimulation with TGF-beta, SMAD proteins become engaged in a multitude of complexes ranging in size from SMAD2-SMAD4 heterodimers to assemblies of >650 kDa. The latter display the highest DNA binding affinity for the TGF-beta-response elements of JUNB and collagen 7. These observations, all validated with endogenous SMAD proteins, modify previous models regarding the assembly and activity of SMAD complexes in the TGF-beta pathway.     

Premature luteinization and cumulus cell defects in ovarian-specific Smad4 knockout mice

SMAD4 is a central component of the TGFbeta superfamily signaling pathway. Within the ovary, TGFbeta-related proteins play crucial roles in controlling granulosa cell growth, differentiation, and steroidogenesis. To study the in vivo roles of SMAD4 during follicle development, we generated an ovarian conditional knockout of Smad4 using the cre/loxP recombination system. Smad4 ovarian-specific knockout mice are subfertile with decreasing fertility over time and multiple defects in folliculogenesis. Regulation of steroidogenesis is disrupted in the Smad4 conditional knockout, leading to increased levels of serum progesterone. In addition, severe cumulus cell defects are present both in vivo and when assayed in vitro. These findings demonstrate that disrupting signaling through SMAD4 in the ovarian granulosa cells leads to premature luteinization of granulosa cells and eventually premature ovarian failure, thereby demonstrating key in vivo roles of TGFbeta superfamily signaling in the timing of granulosa cell differentiation.     

Some Observations on the Characteristics and Distribution of Adenosine Triphosphatases in Young Roots of Pisum sativum, Cultivar Alaska

A study has been made of some characteristics of ATPases fromyoung pea roots and these enzymes have been shown to differfrom ß glycerophosphatases in several respects. DEAEchromotography and gel electrophoresis have confirmed the existenceof a number of different phosphatases with differing affinitiesfor ATP and sodium ß glycerophosphate. At least twoATP-specific phosphatases have been detected and these accountfor about 57 per cent of the total ATPase activity in pea roots. The distribution of ATPase activity along the axis of the roothas been determined by biochemical assay of serial sectionsand by histochemical methods. It is shown that ATPase activityhas a quite different distribution from ß glycero-phosphataseactivity, thus confirming the separate identity of the two enzymesystems. The distribution of ATPases is discussed in relation to theirpossible role in ion transport.     

用免疫共沉淀进一步确定SMAD4中间连接区在形成同源和异源复合物时的作用

用酵母双杂交系统发现 SMAD4的中间连接区能与 SMAD3相互作用 ,而 SMAD4的 N区和 C区不能与 SMAD3相互作用 ,此结果与前人报道的结果有出入 .用细胞免疫共沉淀的方法进一步证实此现象 .结果与酵母双杂交的结果完全吻合 .说明 SMAD4与 SMAD3相互作用形成异源复合物时确实是通过 SMAD4的中间连接区实现的     

Identification of novel protein-protein interactions using a versatile mammalian tandem affinity purification expression system

Identification of protein-protein interactions is essential for elucidating the biochemical mechanism of signal transduction. Purification and identification of individual proteins in mammalian cells have been difficult, however, due to the sheer complexity of protein mixtures obtained from cellular extracts. Recently, a tandem affinity purification (TAP) method has been developed as a tool that allows rapid purification of native protein complexes expressed at their natural level in engineered yeast cells. To adapt this method to mammalian cells, we have created a TAP tag retroviral expression vector to allow stable expression of the TAP-tagged protein at close to physiological levels. To demonstrate the utility of this vector, we have fused a TAP tag, consisting of a protein A tag, a cleavage site for the tobacco etch virus (TEV) protease, and the FLAG epitope, to the N terminus of human SMAD3 and SMAD4. We have stably expressed these proteins in mammalian cells at desirable levels by retroviral gene transfer and purified native SMAD3 protein complexes from cell lysates. The combination of two different affinity tags greatly reduced the number of nonspecific proteins in the mixture. We have identified HSP70 as a specific interacting protein of SMAD3. We demonstrated that SMAD3, but not SMAD1, binds HSP70 in vivo, validating the TAP purification approach. This method is applicable to virtually any protein and provides an efficient way to purify unknown proteins to homogeneity from the complex mixtures found in mammalian cell lysates in preparation for identification by mass spectrometry.