Idiopathic pulmonary arterial hypertension (IPAH) is associated with structural changes to the pulmonary vasculature that ultimately lead to right ventricular failure. A breakthrough in our understanding of the pathogenesis of IPAH was identification in IPAH patients of inactivating heterozygous mutations in the gene encoding the type II bone morphogenetic protein (BMP) receptor (BMPRII). Bone morphogenetic proteins and transforming growth factor (TGF-beta) are a superfamily of polypeptide ligands that transduce signals via their cognate receptors and the Smad family of transcription factors. There are eight different Smads, of which Smads 1, 5 and 8 are BMP receptor-activated Smads. In this study we describe a novel splice-isoform of human Smad8 (Smad8C), and present its preliminary characterization.
Initially, the expression of BMP receptors and their associated Smads in healthy donor lungs and in lungs from patients with IPAH was assessed by reverse-transcription polymerase chain reaction (RT-PCR). No changes in mRNA expression were observed comparing lungs from donors and IPAH patients. However, during the Smad expression studies, a Smad8 splice-variant with a higher molecular mass (which we have called Smad8C) was identified. Sequencing of the full-length Smad8C transcript revealed that the transcript contained an insertion of an additional 111 nucleotides, encoding an additional 37-amino acid residue insertion in the Smad8C polypeptide chain. This additional coding sequence inserted a PY domain (which directs protein stability) into the important regulatory linker region of Smad8, located between the phosphorylation (MH1) and DNA binding (MH2) domains. Unlike all the receptor activated Smads, Smad8C did not exhibit any phosphorylation after BMP or TGF-beta stimulation, but rather dose-dependently inhibited the phosphorylation of endogenous Smad1 upon over-expression of Smad8C in human lung epithelial A549 cells. Furthermore, when Smad8C was over-expressed in A549 cells, the BMP-induced expression of the luciferase gene, when placed downstream of the BRE4 BMP-response element, was inhibited. These data demonstrated that Smad8C could attenuate BMP signaling.
In conclusion, we have identified a novel human Smad8 isoform (Smad8C) containing an additional insertion in its linker region. This novel Smad8 splice isoform appears to negatively-regulate BMP-signaling in lung epithelial cells.
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