Hyperglycemia Regulates RUNX2 Activation and Cellular Wound Healing through the Aldose Reductase Polyol Pathway

Abstract

Diabetes mellitus accelerates cardiovascular microangiopathies and atherosclerosis, which are a consequence of hyperglycemia. The aldose reductase (AR) polyol pathway contributes to these microvascular complications, but how it mediates vascular damage in response to hyperglycemia is less understood. The RUNX2 transcription factor, which is repressed in diabetic animals, promotes vascular endothelial cell (EC) migration, proliferation, and angiogenesis. Here we show that physiological levels of glucose (euglycemia) increase RUNX2 DNA binding and transcriptional activity, whereas hyperglycemia does not. However, inhibition of AR reverses hyperglycemic suppression of RUNX2. IGF-1 secretion and IGF receptor phosphorylation by autocrine IGF-1 occur equally in euglycemic or hyperglycemic conditions, suggesting that reduced RUNX2 activity in response to hyperglycemia is not because of altered IGF-1/IGF receptor activation. AR also negatively regulates RUNX2-dependent vascular remodeling in an EC wounded monolayer assay, which is reversed by specific AR inhibition in hyperglycemia. Thus, euglycemia supports RUNX2 activity and promotes normal microvascular EC migration and wound healing, which are repressed under hyperglycemic conditions through the AR polyol pathway.

NEU1 and NEU3 Sialidase Activity Expressed in Human Lung Microvascular Endothelia

“…HMVEC-Ls and HPAECs were studied in passages 2-7. Human bone marrow ECs (HBMEs) (31) and human umbilical vein retroviral telemorized ECs (Hrvts) were similarly cultured.…”

Hyperglycemia and redox status regulate RUNX2 DNA-binding and an angiogenic phenotype in endothelial cells

“…Hyperglycemia (HG) elevates oxidative stress through the aldose reductase (AR) alternative glucose utilization pathway, which inhibits RUNX2 activity in bone marrow EC (HBME) without altering the levels of total RUNX2 protein (D’Souza et al, 2009). To determine whether this response was conserved in other cells, primary adipose (HAdEC), dermal (HMEC1), and umbilical vein (HRVT) ECs, as well as the classic glucose-responsive cell, the pancreatic β-cell (RINr) were treated with glucose after starvation to lower basal levels of RUNX2 DNA binding.…”

“…Recent findings from our laboratory revealed that RUNX2 is regulated by nutrients in the microenvironment and its DNA-binding activity is especially sensitive to glycemic status (D’Souza et al, 2009). HG inhibited, while anti-oxidants increased, RUNX2 activity and wound healing (D’Souza et al, 2009). Glucose activated RUNX2 DNA binding by increasing phosphorylation through the cdk pathway, which is less active in HG (Pierce et al, 2012).…”

A Quantitative Assay to Study Protein:DNA Interactions, Discover Transcriptional Regulators of Gene Expression, and Identify Novel Anti-tumor Agents

“…In contrast, the quantitative D-ELISA is able to measure the interaction of RUNX2 with DNA-binding sequences corresponding to defined promoter elements in RUNX2 target genes. The use of an anti-RUNX2 antibody increased the specificity of the assay and distinguishes this assay from the traditional gel shift assay 5 . While the D-ELISA is an in vitro assay and cannot survey promoter occupancy in live cells, which is possible with ChIP assays, it can be used to quantitatively screen for compounds that inhibit the DNA-binding complexes.…”

“…Use of an anti-RUNX2 antibody provides specificity to the assay and the lack of radiolabel distinguish this assay from the traditional gel shift assay 5 . Detection of binding complexes is possible with the use of a secondary antibody coupled to horseradish peroxidase (HRP), which converts an HRP substrate, tetramethyl benzidine (TMB) to a colored product for spectrophotometric analysis.…”