Cdc42bpg signaling in arteriosclerosis and vascular fibrosis

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Cardiovascular disease is leading cause of death in the U.S. Assiduous control of blood pressure, cholesterol, and diabetes has yielded significant but incomplete benefit by mitigating atherosclerosis & thromboembolism. Arteriosclerotic conduit vessel stiffening, an overlapping yet distinct disease, also increases risk for stroke, heart failure and amputation risk. No therapies currently treat arteriosclerosis, or the pathological vascular calcification and fibrosis. We've identified that vascular smooth muscle (VSM) parathyroid hormone receptor (PTH1R) signaling limits fibrosis, down regulating transcription driven by hybrid CT(A/T)6GG MEF/SRF cognates in Col3a1 and Cola1 genes. PTH1R restrains mycardin-related transcription factor – a (Mkl1) activation of these elements. We identified that muscular dystrophy protein kinase family members – including ROCK2 & Cdc42 binding proteins (Cdc42bps) – play critical roles in supporting VSM Col3a1 gene expression downstream of PTH1R – mediated inhibition. Cdc42bpg is particularly important - with knockdown almost completely reversing Col3a1 induction occurring with VSM PTH1R deficiency – and is selectively upregulated in aorta by profibrotic angiotensin II (AngII) infusion. Moreover, Cdc42bpg expression significantly stimulates Mkl1-dependent activation of the Col3a1 promoter – the first known, context-informed assay of Cdc42bpg bioactivity. Per Pharos only 8 publications exist on Cdc42bpg, and assays of its functions are rudimentary to non-existent. We propose the following Tasks for this 1-year R03: Task1: Advance and refine our new transcription-based assay of Cdc42bpg activity for systematic domain structure-function analyses. The Cdc42bpg transcriptional regulation assay is deployed in HEK293T cells to identify Cdc42bpg domains necessary to support Col3a1 gene expression via Mkl1. Key observations are independently confirmed in A7r5 aortic VSM. Task2: Establish the Cdc42bpg interactome as regulated by the PTH1R. Cdc42 is unlikely to be the only, or even primary, regulator of Cdc42bpg as relevant to fibrosis. The Cdc42bpg interactome will help identify relevant regulators, providing insights useful for detailed structure-activity refinement. Tandem liquid chromatography – mass spectrometry will identify proteins that co-precipitate following cross-linking with epitope-tagged Cdc42bpg & key subdomains expressed in HEK and A7r5 cells. Focus will be upon those Cdc42bpg interactions that are regulated by PTH1R signals & modulate Col3a1 gene expression. Task3: Study the impact of Cdc42bpg deficiency in AngII - induced cardiovascular fibrosis. The Cdc42bpg-/- knockout mouse (C57BL/6N background) has been generated by KOMP2-BCM, & is being imported to the Towler lab. B6N mice are very susceptible to AngII-induced fibrosis. Thus, arterial, myocardial, and renal fibrosis in the B6N.Cdc42bpg-/- mice will be compared to control B6N animals challenged with AngII infusion in the first in vivo assessment. Cdc42bpg-null alleles will be crossed onto the LDLR-/- background, enabling future studies of Cdc42bpg in diet-induced atherosclerosis, diabetes, & metabolic syndrome.