Pulmonary arterial vascular smooth muscle (PAVSM) cell proliferation is a key pathophysiological component of vascular remodeling in pulmonary arterial hypertension (PAH) for which cellular and molecular mechanisms are poorly understood. The goal of our study was to determine the role of mammalian target of rapamycin (mTOR) in PAVSM cell proliferation, a major pathological manifestation of vascular remodeling in PAH. Our data demonstrate that chronic hypoxia promoted mTOR(Ser-2481) phosphorylation, an indicator of mTOR intrinsic catalytic activity, mTORC1-specific S6 and mTORC2-specific Akt (Ser-473) phosphorylation, and proliferation of human and rat PAVSM cells that was inhibited by siRNA mTOR. PAVSM cells derived from rats exposed to chronic hypoxia (VSM-H cells) retained increased mTOR(Ser-2481), S6, Akt (Ser-473) phosphorylation, and DNA synthesis compared to cells from normoxia-exposed rats. Suppression of mTORC2 signaling with siRNA rictor, or inhibition of mTORC1 signaling with rapamycin and metformin, while having little effect on other complex activities, inhibited VSM-H and chronic hypoxia-induced human and rat PAVSM cell proliferation. Collectively, our data demonstrate that up-regulation of mTOR activity and activation of both mTORC1 and mTORC2 are required for PAVSM cell proliferation induced by in vitro and in vivo chronic hypoxia and suggest that mTOR may serve as a potential therapeutic target to inhibit vascular remodeling in PAH.
Photoreceptor rod cells contain a unique tetraspanin fusion protein known as peripherin/rds. This protein is important in membrane fusion events hypothesized to be essential to disk membrane morphogenesis and disk shedding. In vivo and in vitro fusogenic activity has been mapped to the C-terminal domain of peripherin/rds. Moreover, a fusion peptide domain localized to a 15 amino acid long region (residues 311-325) is essential for mediating lipid bilayer fusion of model membranes. To address the functional and structural properties required for peripherin/rds dependent membrane fusion, constructs of the entire C-terminal domain (residues 284-346) were generated and polypeptides expressed. A wild type-peripherin/rds C-terminal GST fusion construct that included the entire C-terminus (PERCTER) or a C-terminal truncation mutant (PERCTN) were engineered with a thrombin cleavage site. Protein expression was induced in E. coli with IPTG, expressed proteins cleaved from the GST with thrombin and purified to homogeneity on a Superdex 75 column. Purity was confirmed by SDS-PAGE and Western blot analysis. The purified wt C-terminal protein resolved as a monomer under reducing conditions on SDS-PAGE (15%) and was immunoreactive with anti peripherin/rds antibody 2B6 (gift from Dr R. Molday). The purified polypeptide promoted the requisite steps of fusion, membrane destabilization, lipid mixing and aqueous contents mixing. Conversely, the truncation mutant lacking a portion of the fusion domain was unable to promote these steps. A common feature of most membrane fusion proteins is a change in conformation upon membrane association. Structural changes in the C-terminal polypeptide were investigated using far UV CD. The far UV CD spectra of the purified C-terminal polypeptide indicated substantial α-helical content in the wt peptide in isotonic aqueous buffer. An increase in intensity of 208 and 222 nm CD bands upon addition of DPC vesicles indicated an increase in α-helical content of the polypeptide. These results demonstrate that a purified soluble form of the C-terminus of peripherin/rds can interact HHS Public Access Author Manuscript Author ManuscriptAuthor ManuscriptAuthor Manuscript with biological phospholipids; moreover, this interaction promotes a conformational change that is most consistent with an increase in α-helical content.
IRS-1 overexpression has been associated with breast cancer development, hormone independence and antiestrogen resistance. IRS-1 is a major downstream signaling protein for insulin and IGF1 receptors, conveying signals to PI-3K/Akt and ERK1/2 pathways. In estrogen-sensitive breast cancer cell lines, the widely used antiestrogen tamoxifen treatment reduces IRS-1 expression and function, thereby inhibiting IRS-1/PI-3K signaling. IRS-1 may serve as an alternative target to overexpressed IGF1R in breast cancer. While siRNA technology has become commonplace in many laboratories for in vitro gene knockdown studies, and in vivo stability issues are largely solved, its use in vivo is limited by an inability to efficiently and specifically deliver it to the intended site of action. We previously reported reduced survival of human MCF7 estrogen receptor positive breast cancer cells treated with a normal IRS1 siRNA delivered by a cationic lipid, plus an additive effect in combination with tamoxifen. We now report enhanced cellular uptake, relative to the unconjugated serum-stabilized IRS1 siRNA, of a serum-stabilized IRS1 siRNA conjugated with our previously characterized peptide mimetic of IGF1, D-(Cys-Ser-Lys-Cys), without the use of cationic lipids or electroporation, in MCF7 cells that overexpress IGF1R. Excess native IGF1 blocked uptake. An IRS1 siRNA cholesterol conjugate, targeted universally to cell membranes, was taken up by MCF7 cells as much as the peptide mimetic conjugate. IRS1 mRNA knockdown and IRS-1 protein knockdown were comparable for the IGF1 peptide and cholesterol conjugates. The unconjugated serum-stabilized IRS1 siRNA control showed negligible effects. Viability assays showed additive effects of siRNA treatment in combination with tamoxifen. In summary, we have taken the first step in converting an siRNA into a pharmacologically active agent for breast cancer.
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