The activation of MEK/ERK signaling pathway by bone morphogenetic protein 4 to increase hepatocellular carcinoma cell proliferation and migration

Hepatocellular carcinoma (HCC) is one of the most common visceral malignancies worldwide, with a very high incidence and poor prognosis. Bone morphogenesis protein 4 (BMP4), which belongs to the TGF-β superfamily of proteins, is a multifunctional cytokine, which exerts its biologic effects through SMAD- and non-SMAD-dependent pathways, and is also known to be involved in human carcinogenesis. However, the effects of the BMP4 signaling in liver carcinogenesis are not yet clearly defined. Here, we first show that BMP4 and its receptor, BMPR1A, are overexpressed in a majority of primary HCCs and that it promotes the growth and migration of HCC cell lines in vitro. We also establish that BMP4 can induce HCC cyclin-dependent kinase (CDK)1 and cyclin B1 upregulation to accelerate cell-cycle progression. Our study indicates that the induction of HCC cell proliferation is independent of the SMAD signaling pathway, as Smad4 knockdown of HCC cell lines still leads to the upregulation of CDK1 and cyclin B1 expression after BMP4 treatment. Using mitogen-activated protein/extracellular signal-regulated kinase (MEK) selective inhibitors, the induction of CDK1, cyclin B1 mRNA and protein were shown to be dependent on the activation of MEK/extracellular signal-regulated kinase (ERK) signaling. In vivo xenograft studies confirmed that the BMPR1A-knockdown cells were significantly less tumorigenic than the control groups. Our findings show that the upregulation of BMP4 and BMPR1A in HCC promotes the proliferation and metastasis of HCC cells and that CDK1 and cyclin B1 are important SMAD-independent molecular targets in BMP4 signaling pathways, during the HCC tumorigenesis. It is proposed that BMP4 signaling pathways may have potential as new therapeutic targets in HCC treatment.     

Antisense targeting of TGF-beta1 augments BMP-induced upregulation of osteopontin, type I collagen and Cbfa1 in human Saos-2 cells

Despite commonalities in signal transduction in osteoblasts from different species, the role of TGF-beta1 on bone formation remains elusive. In particular, the role of autocrine TGF-beta1 on human osteoblasts is largely unknown. Here we show the effect of TGF-beta1 knock-down on the proliferation and differentiation of osteoblasts induced by BMP2. Treatment with antisense TGF-beta1 moderately increased the rate of cell proliferation, which was completely reversed by the exogenous addition of TGF-beta1. Notably, TGF-beta1 blockade significantly enhanced BMP2-induced upregulation of mRNAs encoding osteopontin, type I collagen and Cbfa1, which was suppressed by exogenous TGF-beta1. Moreover, TGF-beta1 knock-down increased BMP2-induced phosphorylation of Smad1/5 as well as their nuclear import, which paralleled a reduction of inhibitory Smad6. These data suggest autocrine TGF-beta1 antagonizes BMP signaling through modulation of inducible Smad6 and the activity of BMP specific Smad1/5.     

SMAD4-mediated WNT signaling controls the fate of cranial neural crest cells during tooth morphogenesis

TGFβ/BMP signaling regulates the fate of multipotential cranial neural crest (CNC) cells during tooth and jawbone formation as these cells differentiate into odontoblasts and osteoblasts, respectively. The functional significance of SMAD4, the common mediator of TGFβ/BMP signaling, in regulating the fate of CNC cells remains unclear. In this study, we investigated the mechanism of SMAD4 in regulating the fate of CNC-derived dental mesenchymal cells through tissue-specific inactivation of Smad4. Ablation of Smad4 results in defects in odontoblast differentiation and dentin formation. Moreover, ectopic bone-like structures replaced normal dentin in the teeth of Osr2-IresCre;Smad4(fl/fl) mice. Despite the lack of dentin, enamel formation appeared unaffected in Osr2-IresCre;Smad4(fl/fl) mice, challenging the paradigm that the initiation of enamel development depends on normal dentin formation. At the molecular level, loss of Smad4 results in downregulation of the WNT pathway inhibitors Dkk1 and Sfrp1 and in the upregulation of canonical WNT signaling, including increased β-catenin activity. More importantly, inhibition of the upregulated canonical WNT pathway in Osr2-IresCre;Smad4(fl/fl) dental mesenchyme in vitro partially rescued the CNC cell fate change. Taken together, our study demonstrates that SMAD4 plays a crucial role in regulating the interplay between TGFβ/BMP and WNT signaling to ensure the proper CNC cell fate decision during organogenesis.     

Autocrine transforming growth factor-beta signaling mediates Smad-independent motility in human cancer cells

Transforming growth factor-beta (TGF-beta) is a pleiotropic growth factor that plays a critical role in modulating cell growth, differentiation, and plasticity. There is increasing evidence that after cells lose their sensitivity to TGF-beta-mediated growth inhibition, autocrine TGF-beta signaling may potentially promote tumor cell motility and invasiveness. To understand the molecular mechanisms by which autocrine TGF-beta may selectively contribute to tumor cell motility, we have generated MDA-MB-231 breast cancer cells stably expressing a kinase-inactive type II TGF-beta receptor (T beta RII-K277R). Our data indicate that T beta RII-K277R is expressed, can associate with the type I TGF-beta receptor, and block both Smad-dependent and -independent signaling pathways activated by TGF-beta. In addition, wound closure and transwell migration assays indicated that the basal migratory potential of T beta RII-K277R expressing cells was impaired. The impaired motility of T beta RII-K277R cells could be restored by reconstituting TGF-beta signaling with a constitutively active TGF-beta type I receptor (ALK5(TD)) but not by reconstituting Smad signaling with Smad2/4 or Smad3/4 expression. In addition, the levels of ALK5(TD) expression sufficient to restore motility in the cells expressing T beta RII-K277R were associated with an increase in phosphorylation of Akt and extracellular signal-regulated kinase 1/2 but not Smad2. These data indicate that different signaling pathways require different thresholds of TGF-beta activation and suggest that TGF-beta promotes motility through mechanisms independent of Smad signaling, possibly involving activation of the phosphatidylinositol 3-kinase/Akt and/or mitogen-activated protein kinase pathways.     

Role of Smad Proteins in Resistance to BMP-Induced Growth Inhibition in B-Cell Lymphoma

Bone morphogenetic protein (BMP) expression and signaling are altered in a variety of cancers, but the functional impact of these alterations is uncertain. In this study we investigated the impact of expression of multiple BMPs and their signaling pathway components in human B-cell lymphoma. BMP messages, in particular BMP7, were detected in normal and malignant B cells. Addition of exogenous BMPs inhibited DNA synthesis in most lymphoma cell lines examined, but some cell lines were resistant. Tumor specimens from three out of five lymphoma patients were also resistant to BMPs, as determined by no activation of the BMP effectors Smad1/5/8. We have previously shown that BMP-7 potently induced apoptosis in normal B cells, which was in contrast to no or little inhibitory effect of this BMP in the lymphoma cells tested. BMP-resistance mechanisms were investigated by comparing sensitive and resistant cell lines. While BMP receptors are downregulated in many cancers, we documented similar receptor levels in resistant and sensitive lymphoma cells. We found a positive correlation between activation of Smad1/5/8 and inhibition of DNA synthesis. Gene expression analysis of two independent data sets showed that the levels of inhibitory Smads varied across different B-cell lymphoma. Furthermore, stable overexpression of Smad7 in two different BMP-sensitive cell lines with low endogenous levels of SMAD7, rendered them completely resistant to BMPs. This work highlights the role of Smads in determining the sensitivity to BMPs and shows that upregulation of Smad7 in cancer cells is sufficient to escape the negative effects of BMPs.     

Divergence and convergence of TGF-beta/BMP signaling

The transforming growth factor-beta (TGF-beta) superfamily includes more than 30 members which have a broad array of biological activities. TGF-beta superfamily ligands bind to type II and type I serine/threonine kinase receptors and transduce signals via Smad proteins. Receptor-regulated Smads (R-Smads) can be classified into two subclasses, i.e. those activated by activin and TGF-beta signaling pathways (AR-Smads), and those activated by bone morphogenetic protein (BMP) pathways (BR-Smads). The numbers of type II and type I receptors and Smad proteins are limited. Thus, signaling of the TGF-beta superfamily converges at the receptor and Smad levels. In the intracellular signaling pathways, Smads interact with various partner proteins and thereby exhibit a wide variety of biological activities. Moreover, signaling by Smads is modulated by various other signaling pathways allowing TGF-beta superfamily ligands to elicit diverse effects on target cells. Perturbations of the TGF-beta/BMP signaling pathways result in various clinical disorders including cancers, vascular diseases, and bone disorders.     

Transforming growth factor-beta1 downregulation of Smad1 gene expression in rat hepatic stellate cells

Smads are intracellular signaling molecules of the transforming growth factor-beta (TGF-beta) superfamily that play an important role in the activation of hepatic stellate cells (HSCs) and hepatic fibrosis. Excepting the regulation of Smad7, receptor-regulated Smad gene expression is still unclear. We employed rat HSCs to investigate the expression and regulation of the Smad1 gene, which is a bone morphogenetic protein (BMP) receptor-regulated Smad. We found that the expression and phosphorylation of Smad1 are increased during the activation of HSCs. Moreover, TGF-beta significantly inhibits Smad1 gene expression in HSCs in a time- and dose-dependent manner. Furthermore, although both TGF-beta1 and BMP2 stimulate the activation of HSCs, they have different effects on HSC proliferation. In conclusion, Smad1 expression and phosphorylation are increased during the activation of HSCs and TGF-beta1 significantly inhibits the expression of the Smad1 gene.     

Smad1 stabilization and delocalization in response to the blockade of BMP activity

Signaling at the plasma membrane receptors is generally terminated by some form of feedback regulation, such as endocytosis and/or degradation of the receptors. BMP-Smad1 signaling can also be attenuated by BMP-induced expression of the inhibitory Smads, which are negative regulators of Smad1 transactivation activity and/or BMP antagonists. Here, we report on a novel Smad1 regulation mechanism that occurs in response to the blockade of BMP activity. Lowering the serum levels or antagonizing BMPs with noggin led to upregulation of Smad1 at the protein level in several cell lines, but not to upregulation of Smad5, Smad8 or Smad2/3. The Smad1 upregulation occurs at the level of protein stabilization. Upregulated Smad1 was relocalized to the perinuclear region. These alterations seem to affect the dynamics and amplitude of BMP2-induced Smad1 reactivation. Our findings indicate that depleting or antagonizing BMPs leads to Smad1 stabilization and relocalization, thus revealing an unexpected regulatory mechanism for BMP-Smad1 signaling.     

Nodal Signaling Regulates the Bone Morphogenic Protein Pluripotency Pathway in Mouse Embryonic Stem Cells

Members of the transforming growth factor-β superfamily play essential roles in both the pluripotency and differentiation of embryonic stem (ES) cells. Although bone morphogenic proteins (BMPs) maintain pluripotency of undifferentiated mouse ES cells, the role of autocrine Nodal signaling is less clear. Pharmacological, molecular, and genetic methods were used to further understand the roles and potential interactions of these pathways. Treatment of undifferentiated ES cells with SB431542, a pharmacological inhibitor of Smad2 signaling, resulted in a rapid reduction of phosphorylated Smad2 and altered the expression of several putative downstream targets. Unexpectedly, inhibition of the Nodal signaling pathway resulted in enhanced BMP signaling, as assessed by Smad1/5 phosphorylation. SB431542-treated cells also demonstrated significant induction of the Id genes, which are known direct targets of BMP signaling and important factors in ES cell pluripotency. Inhibition of BMP signaling decreased the SB431542-mediated phosphorylation of Smad1/5 and induction of Id genes, suggesting that BMP signaling is necessary for some Smad2-mediated activity. Because Smad7, a known inhibitory factor to both Nodal and BMP signaling, was down-regulated following inhibition of Nodal-Smad2 signaling, the contribution of Smad7 to the cross-talk between the transforming growth factor-β pathways in ES cells was examined. Biochemical manipulation of Smad7 expression, through shRNA knockdown or inducible gene expression, significantly reduced the SB431542-mediated phosphorylation of Smad1/5 and induction of the Id genes. We conclude that autocrine Nodal signaling in undifferentiated mouse ES cells modulates the vital pluripotency pathway of BMP signaling.