Identification And Characterization Of HP, A Novel Protein That Interacts With Smad4

The transforming growth factor-β(TGF-β) signaling plays an important role in cellular differentiation, apoptosis, and proliferation. Smad2, Smad3 and Smad4 proteins are considered to be key mediators of TGF-βsignaling. However, identifications of Smad partners mediating TGF-βsignaling remain rarely reported.Smad4 seems to have a center status in TGF-βsignal transduction pathway. A great quantity of researches indicated that Smad4 plays an important part in the generation, development, and metastasis of malignant tumor. It has been founded that Smad4 induces a large scale of chromatin unfolding by targeting itself to an amplified lac operator-containing chromosome region in mammalian cells, and mapped the unfolding activity at the subdomains within the transactivition domain of Smad4 (260-514aa). By yeast two-hybrid screen, HP, a novel Smad4-interacting protein was isolated from a human mammary library with Smad4 (260-514aa) as bait.To further study the structure, cell location and biological function of HP, the corresponding gene was cloned to construct an eukaryotic expression recombinant plasmid pcDNA3-FLAG-HP. The fact that HP was located in the mammalian cells as a secreted protein was found when the cell lysis and the cell culture supernatant were collected for Western Blot analysis. Furthermore, the recombinant plasmid pcDNA3-HP-enhanced green fluorescent protein (EGFP), expressing HP and EGFP tag, was transfected into MCF-7 cells. The secreting expression process of HP was observed under the fluorescence microscope at different time after transfection.When studying functional activities, it was showed that HP and the deletants can inhibit the transcription of TGF-βresponsive 3TP-luciferase (luc) reporter gene in a TGF-β-independent manner to some extent. The result also showed that HP enhanced c-Myc reporter gene transcriptional activity, meanwhile inhibited PAI-1, P21 transcriptional activity in 293T cells, while the results were opposite in MCF-7 cells with distinct mediation in different cells. In the luc system, the results demonstrated that HP inhibited ERE-Luciferase transcriptional activity, indicating that HP also medicated ER signal transduction by "cross-talk". Subsequently, the expression vectors of HP-small interfering RNAs (siRNAs) were constructed and confirmed by DNA sequencing. Western Blot analysis showed that HP-siRNAs could effectively inhibit the expression of HP gene. It was also discovered that the ability of HP to enhance the transcription of P21 reporter gene was inhibited when MCF-7 cells were co-transfected with HP-siRNAs.Biological functional of HP in cells were further studied. Stable FLAG-HP transfected MCF-7 cell was obtained after G418 resist screening. It was detected by Western Blot that the expressions of P21, P15, P27, plasminogen activator inhibitor-1 (PAI-1), Smad4 and BRCA1 were enhanced and the expressions of c-Myc, c-jun, Rb, Bcl-2 and c-fos proteins were decreased. Meanwhile, MCF-7 cell growth retardation at G2/M was observed by cell flow meter analysis. With viola crystallina assay, HP stable taransfected cells and control were detected to study the influence of HP overexpression on cell growth, and the results showed that MCF-7-HP growed significantly slower than MCF-7-149. Moreover, the conditional medium of HP-MCF-7 and 149-MCF-7 was collected to study the influence of secreted HP protein on cell growth. The results indicated that one, two and three days cultured conditional medium can inhibit the growth of breast cancer, lung cancer, hepatoma cancer cells at distinct levels; while the inhibition was not obvious in normal cells, including normal breast cells, lung cells, hepatoma cells, fibroblastic cells and SV40 transfromed 293T cells.TβRⅡinhibitor SB431542 was added to explore whether HP contact with TβR in signal transduction pathway. No obvious difference was obtained in Western Blot analysises at different times and different doses of SB431542 treated protein expression, including P21,PAI-1,c-Myc. The result indicated that secreted HP protein did not contact with TβRⅡbut possibly with ligands to activate TβRⅠto mediate the signal pathway. Implication: these results identified a new regulating factor, HP, which plays a role in TGF-βsignaling pathway through interaction with Smad4. Further study will be helpful to understand its specific regulating mechanism in TGF-βsignaling pathway.