The long term objective of this research proposal is to determine the molecular mechanisms of vascular calcification in order to identify novel target(s) for the treatment of chronic kidney disease (CKD)-dependent vascular calcification. Vascular calcification is closely associated with cardiovascular morbidity and mortality in patients with CKD. In fact, more than half of all deaths in CKD subjects can be attributed to cardiovascular diseases. We hypothesized that a central event in the pathogenesis of CKD-dependent vascular calcification is increased expression of phosphorylated activating transcription factor 4 (ATF4). ATF4 is a member of the cAMP-responsive element-binding protein (CREB) family of basic zipper-containing transcription factors that regulates osteogenesis and also mediates unfolded protein response (UPR) in the endoplasmic reticulum (ER). Our hypothesis is based on the following evidence derived from a series of preliminary results from our lab: 1) total and phosphorylated ATF4 protein levels were induced by a number of positive regulatory factors for vascular calcification, such as inorganic phosphate, inflammatory cytokines (TNFa) and saturated fatty acids through the activation of the PERK-elF2a axis of the UPR in vascular smooth muscle cells (VSMCs); 2) adenovirus-mediated overexpression of ATF4 induced mineralization of VSMCs; 3) ATF4 knockdown, on the other hand, attenuated vascular calcification; 4) serine-phosphorylation of ATF4 (p-ATF4) was induced by PKA activation by forskolin, which is known to promote vascular calcification; 5) PKA and ERK inhibitors inhibited the phosphorylation of ATF4, resulting in the reduction of vascular calcification; 6) Total ATF4 and p-ATF4 proteins were increased in the aortas of murine models of atherosclerotic calcification such as ApoE knockout mice with 5/6 nephrectomy (5/6 nx), and medial calcification such as DBA2/J mice with 5/6 nx and klotho knockout mice; 7) ATF4 targets (CHOP and GADD34) increased in these models and 8) ATF4 regulates the expression of Pit-1, a major phosphate transporter in VSMCs. To determine the pivotal role of ATF4 in the pathogenesis of vascular calcification, we propose three specific aims. Specific Aim 1: Determine whether global ATF4 deficiency and overexpression modulate CKD-dependent medial and atherosclerotic calcification. Specific Aim 2: Determine whether VSMC-specific activation and inhibition of ATF4 influence CKD-dependent medial and atherosclerotic calcification. Specific Aim 3: Elucidate molecular mechanisms by which ATF4 regulates osteoblastic differentiation and mineralization of VSMCs.

R01DK096030

2013-04-01

2019-03-31