Elsevier

Cellular Signalling

Volume 15, Issue 2, February 2003, Pages 181-187
Cellular Signalling

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

https://doi.org/10.1016/S0898-6568(02)00088-8Get rights and content

Abstract

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-β 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 p21CIP-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.

Introduction

Growth/differentiation factor-5 (GDF-5) is a member of the bone morphogenetic protein (BMP) family which, in turn, belongs to the transforming growth factor-β (TGF-β) superfamily [1], [2]. GDF-5 is expressed in a variety of tissues, most prominently in early developing limbs, and has been implicated in neurotrophic functions [3], [4]. It is also known that GDF-5 induces chondroblastic and osteoblastic differentiation in clonal cell lines and primary cultures [5], [6]. Recently, GDF-5 was found to enhance angiogenesis in both chick chorioallantoic membrane and rabbit cornea assays [7]. In addition to the activities shown above, TGF-β superfamily members are involved in the regulation of many biological processes in mesenchymal as well as other tissues. We previously reported that activin A and BMP-2, TGF-β superfamily members, induce cell cycle arrest in the G1 phase and apoptosis in mouse B lineage cells and myeloma cells [8], [9].

TGF-β superfamily members elicit their multifunctional effects through heteromeric complexes of types I and II serine/threonine kinase receptors [10]. Furthermore, members of the Smad protein family are known to play pivotal roles in intracellular TGF-β family signalling, and the activated type I receptor propagates the signal through transient interaction with and phosphorylation of receptor-restricted Smads [11]. Thus, Smad1, Smad2, Smad3 and Smad5 become phosphorylated by specific activated type I serine/threonine kinase receptors and act in a pathway-restricted fashion [12]. Smad4 forms hetero-oligomeric complexes with pathway-restricted Smad proteins, which then translocate into the nucleus and activate transcriptional responses [13], [14].

Smad6 and Smad7 function as inhibitors of TGF-β superfamily signaling [15], [16], and the latter forms stable interactions with the activated TGF-β type I receptor, thereby preventing the activation of receptor-restricted Smads because of competition for receptor binding. Furthermore, Smad7 has been shown to bind and inhibit signalling downstream of BMP receptors [16]. In contrast, Smad6 specifically blocks BMP signalling, but not that of activin, in mink lung epithelial cells [17]. Smad6 has also been shown to bind to BMP receptors and inhibit signalling downstream of the receptors [15], and to prevent formation of an active Smad4/Smad1 signalling complex by directly competing with Smad1 for binding to Smad4 [17]. Thus, Smad7 may act as a general inhibitor against TGF-β superfamily members and Smad6 may have a tendency preferentially to inhibit BMP signalling.

We previously demonstrated that both Smad6 and Smad7 are BMP-2-inducible antagonists of BMP-2-induced growth arrest and apoptosis in mouse B cell hybridoma HS-72 cells [18]. We have also reported that activin A and BMP-2 induce cell cycle arrest in the G1 phase and apoptosis in HS-72 cells [19]. However, there are no known reports of the precise mechanism by which GDF-5 exerts its negative growth effect on B lineage cells. In the present study, we found that another BMP-2 family member, GDF-5, induces cell cycle arrest and apoptosis in B lineage cells with modulation by Smad proteins.

Section snippets

Reagents and antibodies

Recombinant GDF-5, recombinant BMP-2 and activin A were kindly supplied by BIOPHARM (Heidelberg, Germany), Yamanouchi Pharmaceutical (Tokyo, Japan) and Ajinomoto (Tokyo, Japan), respectively. Anti-p21CIP-1/WAF-1 (F-5) and anti-retinoblastoma protein (Rb) (G3-245) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and Pharmingen (San Diego, CA), respectively.

Cell culture

HS-72, a mouse hybridoma cell line, was maintained in Iscove's Modified Dulbecco's medium (IMDM; GIBCO BRL, Grand

GDF-5 induces growth arrest of HS-72 cells

BMP-2, BMP-4, BMP-7 and GDF-5, each a member of the BMP family, have been reported to enhance bone healing and remodelling [22]. These molecules were initially isolated because of their remarkable activity to induce cartilage and bone formation at ectopic sites in animals [23], and subsequently, many additional BMP members have been identified based on their sequence homology. Among them, the Gdf5 gene product, GDF-5, has been identified as belonging to a subgroup of the BMP family, which

Acknowledgements

We thank Dr. Jens Pohl (BIOPHARM) for providing the GDF-5, Dr. Eto (Ajinomoto) for the activin A and Yamanouchi Pharmaceutical for the BMP-2. This study was supported in part by Grants in Aid for Scientific Research (nos. 13671906 and 14657533) from the Ministry of Education, Science and Culture of Japan.

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