Growth/differentiation factor-5 induces growth arrest and apoptosis in mouse B lineage cells with modulation by Smad

Bone morphogenetic proteins, including growth/differentiation factor-5 (GDF-5), are multifunctional cytokines. Recent studies of intracellular signal transduction mechanisms for the transforming growth factor-beta superfamily have focused on Smad proteins. However, scant attention has been given to the mechanism by which GDF-5 exerts its negative growth effect on immunological competent cells. In the present study, we demonstrated that GDF-5 induced cell cycle arrest in the G1 phase before the appearance of apoptosis in mouse B cell hybridoma HS-72 cells, while the ectopic expression of Smad6 and Smad7 in HS-72 cells suppressed the GDF-5-induced G1 cell cycle arrest by abolishing the expression of p21(CIP-1/WAF-1) and hypophosphorylation of retinoblastoma protein. Moreover, we found that Smad6 and Smad7 suppressed GDF-5-induced apoptosis in HS-72 cells. These findings indicated that Smad6 and Smad7 exhibit inhibitory effects toward GDF-5-mediated signaling in B lineage cells.     

The Role of Activin Type I Receptors in Activin A-Induced Growth Arrest and Apoptosis in Mouse B-Cell Hybridoma Cells

Activins transduce their signals by binding to activin type I receptors and activin type II receptors, both of which contain a serine/threonine kinase domain. In this study, we established stable transfectants expressing two types of activin receptors, ActRI and ActRIB, to clarify the role of these receptors in activin signalling for growth inhibition in HS-72 mouse B-cell hybridoma cells. Over-expression of ActRI suppressed activin A-induced cell-cycle arrest in the G1 phase caused by inhibition of retinoblastoma protein phosphorylation through induction of p21^CIP1/WAF1, a cyclin-dependent kinase inhibitor, and subsequent apoptosis. In contrast, HS-72 clones that over-expressed ActRIB significantly facilitated activin A-induced apoptosis. These results indicate that ActRI and ActRIB are distinct from each other and that the ActRI/ActRIB expression ratio could regulate cell-cycle arrest in the G1 phase and subsequent apoptosis in HS-72 cells induced by activin A.     

Intracellular signaling of osteogenic protein-1 through Smad5 activation

Smad proteins play pivotal roles in the intracellular signaling of the multifunctional transforming growth factor-β (TGF-β) family members downstream of serine/threonine kinase type I and type II receptors. Smad2 and Smad3 are specific mediators of TGF-β and activin, while Smad1 and Smad5 are involved in bone morphogenetic protein-2 (BMP-2) and BMP-4 signaling. Here we report that osteogenic protein-1 (OP-1), also termed BMP-7, binds predominantly to BMPR-IB in the rat osteoprogenitor-like cell line, ROB-C26. Smad1, Smad5, and Smad8, but not Smad2 and Smad3, were found to stably interact with the kinase-deficient BMPR-IB after it was phosphorylated by the BMPR-II kinase. In ROB-C26 cells, which express Smad2, Smad3, Smad4, and Smad5, OP-1 was found to stimulate the phosphorylation of Smad5. Whereas transfection of wild-type Smad5 enhanced the OP-1-induced response, transfection of wild-type Smad2 had no effect on OP-1 signaling. A Smad5-2SA mutant, in which the two most carboxy-terminal serine residues were mutated to alanine residues, was found to act as a dominant negative inhibitor of OP-1-induced responses upon its transfection into various cell types, including ROB-C26 cells, in contrast to ectopic expression of a Smad2-2SA mutant which was without effect. Smad5, therefore, is a key component in the intracellular signaling of OP-1. J. Cell. Physiol. 177:355–363, 1998. © 1998 Wiley-Liss, Inc.     

Dissociation of bone morphogenetic protein-mediated growth arrest and apoptosis of mouse B cells by HPV-16 E6/E7

We have previously found that bone morphogenetic protein-2 (BMP-2), a member of the transforming growth factor-beta family, induces cell-cycle arrest in the G1 phase and apoptotic cell death of HS-72 mouse hybridoma cells. In this study, we show that BMP-2 did not alter expression of cyclin D, cyclin E, cyclin-dependent kinase 2 (CDK2), CDK4, p27KIP1, p16INK4a, or p15INK4b, but enhanced expression of p21(CIP1/WAF1). Accumulation of p21(CIP1/WAF1) resulted in increased binding of p21(CIP1/WAF1) to CDK4 and concomitantly caused a profound decrease in the in vitro retinoblastoma protein (Rb) kinase activity of CDK4. Furthermore, the ectopic expression of human papilloma virus type-16 E7, an inhibitor of p21(CIP1/WAF1) and Rb, reverted G1 arrest induced by BMP-2. Expression of E6/E7, without increasing the p53 level, blocked inhibition of Rb phosphorylation and G1 arrest, but did not attenuate cell death in BMP-treated HS-72 cells. Taken together, these results suggest that inhibition of Rb phosphorylation by p21(CIP1/WAF1) is responsible for BMP-2-mediated G1 arrest and that BMP-2-induction of apoptosis might be independent of Rb hypophosphorylation.     

Smad7 is induced by norepinephrine and protects rat hepatocytes from activin A-induced growth inhibition

Activin A induces growth arrest of rat hepatocytes in vitro and in vivo. The alpha(1)-adrenergic agonist, norepinephrine (NE), enhances epidermal growth factor-stimulated DNA synthesis and inhibits activin A-induced growth inhibition, but the mechanisms of these actions are unclear. Smad proteins have recently been identified as intracellular signaling mediators of transforming growth factor-beta family members. In the present study, we explored how NE modulates the Smad signaling pathway in rat cultured hepatocytes. We demonstrate that NE inhibits activin A-induced nuclear accumulation of Smad2/3 and that NE rapidly induces inhibitory Smad7 mRNA expression. Infection of Smad7 adenovirus into rat hepatocytes inhibited activin A-induced nuclear accumulation of Smad2/3, enhanced epidermal growth factor-stimulated DNA synthesis, and abolished the growth inhibitory effect of activin A. We also demonstrated that the induction of Smad7 by NE is dependent on nuclear factor-kappa B (NF-kappa B). The amount of active NF-kappa B complex rapidly increased after NE treatment. Preincubation of the cells with an NF-kappa B pathway inhibitor N-tosyl-l-phenylalanine chloromethyl ketone or infection of the cells with an adenovirus expressing an I kappa B super-repressor (Ad5I kappa B) abolished the NE-induced Smad7 expression. These results indicate a mechanism of transmodulation between the Smad and trimeric G protein signaling pathways in rat hepatocytes.     

BMP2-induced apoptosis is mediated by activation of the TAK1-p38 kinase pathway that is negatively regulated by Smad6

Bone morphogenetic protein 2 (BMP2), a member of the transforming growth factor-beta (TGF-beta) superfamily, regulates a variety of cell fates and functions. At present, the molecular mechanism by which BMP2 induces apoptosis has not been fully elucidated. Here we propose a BMP2 signaling pathway that mediates apoptosis in mouse hybridoma MH60 cells whose growth is interleukin-6 (IL-6)-dependent. BMP2 dose-dependently induces apoptosis in MH60 cells even in the presence of IL-6. BMP2 has no inhibitory effect on the IL-6-induced tyrosine phosphorylation of STAT3, and the bcl-2 gene expression which is known to be regulated by STAT3, suggesting that BMP2-induced apoptosis is not attributed to alteration of the IL-6-mediated bcl-2 pathway. We demonstrate that BMP2 induces activation of TGF-beta-activated kinase (TAK1) and subsequent phosphorylation of p38 stress-activated protein kinase. In addition, forced expression of kinase-negative TAK1 in MH60 cells blocks BMP2-induced apoptosis. These results indicate that BMP2-induced apoptosis is mediated through the TAK1-p38 pathway in MH60 cells. We also show that MH60-derived transfectants expressing Smad6 are resistant to the apoptotic signal of BMP2. Interestingly, this ectopic expression of Smad6 blocks BMP2-induced TAK1 activation and p38 phosphorylation. Moreover, Smad6 can directly bind to TAK1. These findings suggest that Smad6 is likely to function as a negative regulator of the TAK1 pathway in the BMP2 signaling, in addition to the previously reported Smad pathway.     

Methylation of Smad6 by protein arginine N-methyltransferase 1

Signal transduction pathways utilize posttranslational modifications to regulate the activity of their components in a temporal-spatial and efficient fashion. Arginine methylation is one of the posttranslational modifications that can result in monomethylated-, asymmetric dimethylated- and/or symmetric dimethylated-arginine residues in proteins. Here we demonstrate that inhibitory-Smads (Smad6 and Smad7), but not receptor-regulated- (R-)Smads and the common-partner Smad4, can be methylated by protein arginine N-methyltransferase (PRMT)1. Using mass-spectrometric analysis, we found that PRMT1 dimethylates arginine(74) (Arg(74)) in mouse Smad6. PRMT1 interacts with the N-terminal domain of Smad6 in which Arg(74) residue is located. Assays examined so far have shown no significant differences between the functions of Smad6 and those of methylation-defective Smad6 (Smad6R74A). Both wild-type and Smad6R74A were equally efficient in blocking BMP-induced growth arrest upon their ectopic expression in HS-72 mouse B-cell hybridoma cells.     

Smad7-deficient mice show growth retardation with reduced viability

Smad7 is an inhibitory molecule induced by members of the transforming growth factor-β (TGF-β) family, including TGF-β, activin, nodal and bone morphogenetic proteins (BMPs). To elucidate the in vivo functions of Smad7, we generated conditional Smad7-knockout mice in which the Mad homology 2 (MH2) domain and the poly (A) signal sequence were flanked with loxP sites (floxed). The Smad7-floxed mice exhibited no obvious phenotype. Smad7 total-null mice on a C57BL/6 background died within a few days of birth, whereas mice with an ICR background developed to adulthood but were significantly smaller than wild-type mice. Unexpectedly, phospho-Smad2 and phospho-Smad3 were decreased in Smad7-deficient mouse embryonic fibroblast (MEF) cells, whereas phospho-Smad1/5/8 was similarly expressed in wild-type and Smad7-deficient MEF cells. Moreover, expression levels of TGF-β type I receptor (ALK5) were higher in Smad7-deficient MEF cells than in wild-type MEF cells. Plasminogen activator inhibitor-1 (PAI-1) and inhibitor of differentiation-1 (Id-1) mRNA were similarly expressed in wild-type and Smad7-deficient MEF cells. Some differences were observed in mitogen-activated protein kinase (MAPK)-signalling between wild-type and Smad7-deficient MEF cells. We demonstrated that Smad7 plays an important role in normal mouse growth and provide a useful tool for analysing Smad7 functions in vivo.     

Selective inhibitory effects of Smad6 on bone morphogenetic protein type I receptors

The inhibitory Smads, Smad6 and Smad7, play pivotal roles in negative regulation of transforming growth factor-beta (TGF-beta) family signaling as feedback molecules as well as mediators of cross-talk with other signaling pathways. Whereas Smad7 acts as a ubiquitous inhibitor of Smad signaling, Smad6 has been shown to effectively inhibit bone morphogenetic protein (BMP) signaling but only weakly TGF-beta/activin signaling. In the present study, we have found that Smad6 inhibits signaling from the activin receptor-like kinase (ALK)-3/6 subgroup in preference to that from the ALK-1/2 subgroup of BMP type I receptors. The difference is attributable to the interaction of Smad6 with these BMP type I receptors. The amino acid residues responsible for Smad6 sensitivity of ALK-3 were identified as Arg-238, Phe-264, Thr-265, and Ala-269, which map to the N-terminal lobe of the ALK-3 kinase domain. Although Smad6 regulates BMP signaling through multiple mechanisms, our findings suggest that interaction with type I receptors is a critical step in the function of Smad6.