Betaglycan (TβRIII) Is Expressed in the Thymus and Regulates T Cell Development by Protecting Thymocytes from Apoptosis

et al. (2012) Betaglycan (TbRIII) Is Expressed in the Thymus and Regulates T Cell Development by Protecting Thymocytes from Apoptosis. PLoS ONE 7(8): e44217. doi:10.1371/journal.pone.0044217 Betaglycan (TbRIII) Is Expressed in the Thymus and Regulates T Cell Development by Protecting Thymocytes from Apoptosis German R. Aleman-Muench 0 Valentin Mendoza 0 Kaye Stenvers 0 Eduardo A. Garcia-Zepeda 0 Fernando Lopez-Casillas 0 Chander Raman 0 Gloria Soldevila 0 Jean Kanellopoulos, University Paris Sud, France 0 1 Departamento de Inmunolog a, Instituto de Investigaciones Biome dicas, Universidad Nacional Auto noma de Me xico, Me xico, Me xico, 2 Departamento de Biolog a Celular, Instituto de Fisiolog a Celular, Universidad Nacional Auto noma de Me xico, Me xico, Me xico, 3 Reproductive Development and Cancer laboratory, Prince Henry9s Institute of Medical Research , Clayton, Victoria , Australia , 4 Departments of Medicine and Microbiology, Division of Clinical Immunology and Rheumatology University of Alabama at Birmingham , Alabama , United States of America TGF-b type III receptor (TbRIII) is a coreceptor for TGFb family members required for high-affinity binding of these ligands to their receptors, potentiating their cellular functions. TGF-b [1-3], bone morphogenetic proteins (BMP2/4) and inhibins regulate different checkpoints during T cell differentiation. Although TbRIII is expressed on hematopoietic cells, the role of this receptor in the immune system remains elusive. Here, we provide the first evidence that TbRIII is developmentally expressed during T cell ontogeny, and plays a crucial role in thymocyte differentiation. Blocking of endogenous TbRIII in fetal thymic organ cultures led to a delay in DN-DP transition. In addition, in vitro development of TbRIII2/2 thymic lobes also showed a significant reduction in absolute thymocyte numbers, which correlated with increased thymocyte apoptosis, resembling the phenotype reported in Inhibin a 2/2 thymic lobes. These data suggest that Inhibins and TbRIII may function as a molecular pair regulating T cell development. - Funding: Work in GSs lab was supported by a grant from DGAPA, UNAM (PAPIIT #IN228205). GRAM was a recipient of a doctoral fellowship #208213 from CONACyT. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. T cell development requires the recognition of self-peptide MHC complexes by immature thymocytes, leading to the selection of a self-restricted and autotolerant T cell repertoire. In addition to the nature of TCR signals triggered by self-peptide recognition, other signals provided by thymic stromal cells, such as those triggered by members of TGF-b superfamily like TGFb, activins/ inhibin and BMP subfamilies have been shown to act as key regulators of apoptosis, survival and cell cycle progression in different cell types [13]. We and others have described that members of TGF-b superfamily are differentially expressed in the thymus and regulate specific developmental checkpoints, influencing T cell development in [4,5]. Specifically, among TGFbs, only TGFb1 and TGFb2 appear to regulate DN1-DN2 and DN-DP transitions and promote maturation of CD8SP [4]. On the other hand, BMPs and their negative regulators, chordin, noggin and twisted gastrulation (Tsg), are also expressed in the thymus. BMP2 and BMP4 were shown to negatively regulate DN1-DN2, DN3-DN4 and DN-DP transitions [4,5]. Finally, we have recently described that inhibins are abundantly expressed in the thymus by stromal cells and thymocytes [6] and that, addition of exogenous inhibins in FTOCs regulate T cell development at the DN3-DN4, DN-DP, and DPCD8SP stages [7]. Moreover, endogenous inhibins were required to obtain normal thymocyte numbers and adequate DN-DP transition during in vitro T cell development [7]. A central coreceptor in the canonical signaling pathway of TGF-b is betaglycan, also known as the TGF-b type III receptor (TbRIII), which is a widely expressed membrane-anchored proteoglycan. Structurally, it is characterized by a large extracellular region, containing heparan and chondroitin sulphate chains, and a short cytoplasmic domain that lacks a signaling motif, which has recently been shown to regulate cell processes like apoptosis and cell migration [8,9]. TbRIII-null mice embryos die between E13.5 to E18.5 of embryonic stage by heart and liver defects, caused by an altered TGF-b2-induced mesenchymal transformation process and the incidence of apoptotic events [10]. Recently, it has been described that the absence of TbRIII also compromises normal seminiferous cord formation, Leydig cell function in testis [11] and alters kidney development [12]. The main function of TbRIII is to orchestrate the TGFb, BMP and inhibin-mediated signals in different cell types. TbRIII enhances the binding of all three TGF-b isoforms to the TGF-b signaling receptor complex, but is specially required for high affinity binding and functional activity of TGF-b2 [10,1317]. On the other hand, TbRIII enhances the binding of BMP2, BMP4, BMP7 and GDF5 to BMPR1 leading to an increase in Smad1 phosphorylation, and thus potentiates its functional effects [18]. In addition, TbRIII binds inhibins with high affinity, potentiating the exclusion of ACTRIB (ALK4) to antagonize activin-mediated functions [14,1921]. Also, TbRIII enables inhibins to antagonize BMP signaling [22] and to reduce TGF-b2 signals through the endocytic internalization of TbRIII [23]. Conversely, TbRIII also allow TGF-b1 and 2 to attenuate inhibin-mediated functions by downregulating the expression and binding of this co-receptor [24,25]. Although TbRIII is broadly expressed in many tissues, its presence and the potential function of this receptor in the hematopoietic system remains poorly characterized [2631]. Given that downstream signaling of many TGF-b ligands are regulated by TbRIII to fine tune key cellular processes, here we investigated the expression of TbRIII in the thymus and its potential role in T cell differentiation. Materials and Methods Mice 4 to 6 week old C57BL/6 mice were used in our experiments. TbRIII wild type, heterozygous, and null mouse embryos [10] were obtained from synchronized embryonic day 14 (E14) matings of TbRIII heterozygous mice. All animal handling and experimental procedures were done according to the Instituto de Investigaciones Biomedicas ethics guidelines. The study was approved by the Comite para el Cuidado y Uso de Animales de Laboratorio (CCUAL) of the Institute. PCR and Genotyping Amplification of wild-type or mutant TbRIII alleles in tissue samples from E14 embryos was achieved with the following primers: P1 59-ATTGTGTTCATAGGTCCAGA-39, wt 5CCTAGTCCTTGGTCTGTACT-39, and Neo 59TAGGGTTCCGATTTAGTGCT- 39, using the following program: 1 minute of initial denaturing step at 94uC, then 3 (...truncated)