Molecular Basis for Dysregulated Activation of NKX2‐5 in the Vascular Remodeling of Systemic Sclerosis

Abstract

Objective NKX2‐5 is a homeobox transcription factor that is required for the formation of the heart and vessels during development, with significant postnatal down‐regulation and reactivation in disease states, characterized by vascular remodeling. The purpose of this study was to investigate mechanisms that activate NKX2‐5 expression in diseased vessels, such as systemic sclerosis (scleroderma; SSc)–associated pulmonary hypertension (PH), and to identify genetic variability that potentially underlies susceptibility to specific vascular complications.MethodsWe explored NKX2‐5 expression in biopsy samples from patients with SSc‐associated PH and in pulmonary artery smooth muscle cells (PASMCs) from patients with scleroderma. Disease‐associated putative functional single‐nucleotide polymorphisms (SNPs) at the NKX2-5 locus were cloned and studied in reporter gene assays. SNP function was further examined through protein–DNA binding assays, chromatin immunoprecipitation assays, and RNA silencing analyses.ResultsIncreased NKX2‐5 expression in biopsy samples from patients with SSc‐associated PH was localized to remodeled vessels and PASMCs. Meta‐analysis of 2 independent scleroderma cohorts revealed an association of rs3131917 with scleroderma (P = 0.029). We demonstrated that disease‐associated SNPs are located in a novel functional enhancer, which increases NKX2-5 transcriptional activity through the binding of GATA‐6, c‐Jun, and myocyte‐specific enhancer factor 2C. We also characterized an activator/coactivator transcription‐enhancer factor domain 1 (TEAD1)/Yes‐associated protein 1 (YAP1) complex, which was bound at rs3095870, another functional SNP, with TEAD1 binding the risk allele and activating the transcription of NKX2-5.Conclusion NKX2-5 is genetically associated with scleroderma, pulmonary hypertension, and fibrosis. Functional evidence revealed a regulatory mechanism that results in NKX2-5 transcriptional activation in PASMCs through the interaction of an upstream promoter and a novel downstream enhancer. This mechanism can act as a model for NKX2‐5 activation in cardiovascular disease characterized by vascular remodeling.

SNPs in Sites for DNA Methylation, Transcription Factor Binding, and miRNA Targets Leading to Allele-Specific Gene Expression and Contributing to Complex Disease Risk: A Systematic Review

“…Vascular remodeling is the structural rearrangement of blood vessels’ walls in response to repair and inflammation [98]. Vascular remodeling is the hallmark for pulmonary arterial hypertension, atherosclerosis, and scleroderma [98].…”

Metformin intervention prevents cardiac dysfunction in a murine model of adult congenital heart disease

“…Preliminary analysis of hematocrit in ACHD adult mice has shown no significant differences in blood cell composition (data not shown). It is also possible de novo expression of Nkx2-5 in disease states modifies the cardiovascular phenotype, as seen in atherosclerotic plaques in ApoE–deficient mice and patients with pulmonary hypertension [50], [51]. However, mice do not normally develop atherosclerosis.…”

Pulmonary arterial hypertension: Cellular and molecular changes in the lung

“…Under conditions of oxidant stress akin to PAH, NEDD9 accumulation was observed in PAECs owing to disruption in NEDD9-SMAD3 complex formation, which is required for normal proteasomal degradation of NEDD9. Increased bioavailable NEDD9, in turn, interacts with the cardiac developmental transcription factor NKx2-5 31 to directly upregulate COL3A1 transcription, increase collagen III expression, and induce cellular stiffening in PAECs. Furthermore, increased NEDD9 was implicated in paracrine cross-talk via exosome signaling between PAECs and other cell types with fibrotic potential, suggesting that PAECs may function as a portal of entry into a wider cellular fibrotic program.…”