High glucose and/or high insulin affects HIF-1 signaling by regulating AIP1 in human umbilical vein endothelial cells
Introduction
Diabetes mellitus is a chronic disease threatening the health of individuals worldwide [1]. Researchers have attempted to elucidate the molecular mechanism of impaired functioning of cells during diabetes and to identify novel targeted therapies. Apoptosis signal-regulating kinase 1-interacting (ASK1-interacting) protein-1 (AIP1), also known as DAB2-interacting protein (DAB2IP), which is a member of the Ras GTPase-activating protein family, has been implicated in cell growth inhibition and apoptosis [2], [3]. AIP1, via its Ras GTPase-activating protein (Ras-GAP) activity, inhibits Ras-mediated cell survival signaling, resulting in cell growth inhibition [4]. However, AIP1 functions as a positive regulator of apoptosis by mediating activation of the apoptotic kinase apoptosis signal-regulating kinase 1 (ASK1) [3]. Consistent with its role as an inhibitor of cell survival and growth, AIP1 expression is often downregulated in various human cancers [2], [5], [6]. Recently, it has been reported that AIP1 is recruited to the VEGFR2-PI3K complex, binding to both VEGFR2 and PI3K p85 during a late phase of the VEGF response, and that this activity leads to the inhibition of VEGFR2 signaling. Considering the critical role of VEGFR2 signaling in angiogenesis, the regulation of VEGFR2 activity/activation may represent an important mechanism for the control of angiogenesis [7]. Studies have suggested that AIP1 may function as a tumor suppressor gene. However, the effects of high glucose and insulin on AIP1 gene expression are still unknown.
Hypoxia-inducible factor-1 (HIF-1), which is a key transcription factor, is known to promote tissue vascularization by activating the transcription of genes encoding angiogenic factors [8]. HIF-1 is a heterodimer composed of the rate-limiting factor, HIF-1α, and constitutively expressed HIF-1β [9], [10]. During hypoxia, the regulatory subunit of the HIF-1 heterodimer, HIF-1α, is stabilized through a decrease in the activity of prolyl hydroxylases that target the protein for degradation [11]. The regulation of HIF-1α can also occur independent of the oxygen level in the environment. Under normoxia, HIF-1α can be activated by the phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR or mitogen-activated protein kinase (MAPK) pathways initiated by growth factors, cytokines, and other signaling molecules [12], [13], [14]. Studies have demonstrated that PI3K/Akt signaling is required for VEGF expression through HIF-1 in response to growth factor stimulation and oncogene activation [12], [15]. Previous studies have shown that glucose and hyperglycemia impair the stability and function of HIF-1α [16], [17]. However, the molecular mechanisms underlying this impairment in HIF-1 signaling and its relation to AIP1 regulation have not been elucidated.
To define the roles of AIP1 and HIF-1 signaling in diabetes, we detected changes in AIP1 expression and HIF-1 signaling in cells exposed to high glucose, high insulin, and the combination of the two (high glucose + high insulin), which simulate conditions observed during the progression of type 2 diabetes. Therefore, we hypothesized that the inhibition of HIF-1 signaling activity in human umbilical vein endothelial cells observed in diabetes is a result of the regulation of AIP1.
Section snippets
Materials
Human umbilical vein endothelial cells (HUVECs) were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). Endothelial cell growth medium 199 (M199) and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY, USA). Endothelial growth factor (EGF) was purchased from PeproTech Asia (Rehovot, Israel). Lipofectamine RNAiMAX Reagent was purchased from Invitrogen Canada (Burlington, ON, Canada). TriPure and SYBR Green I fluorescent dyes were purchased from
High glucose stimulates AIP1 expression and decreases HIF-1α signaling
The results showed that 30 mmol/L glucose (HG) alone or the combination of high glucose and high insulin (HG/HI, 1 nmol/L insulin) increased AIP1 expression at the mRNA and protein levels accompanied by decreases in HIF-1α and VEGF compared with the LG group (Fig. 1A). AIP1 expression in the cells under high glucose showed 2.47-fold (P = 0.000) and 8.78-fold (P = 0.000) increases at the mRNA and protein levels, respectively, compared with those of cells under low glucose (HG/HI: 1.62-fold (P = 0.026)
Discussion
The mechanism underlying the disordered signaling and impaired functioning of cells during diabetes is still unclear. The experimental conditions of high glucose, high insulin, and high glucose + high insulin were designed to recapitulate the successive phases of type 2 diabetes. Emerging data suggest that each of these phases may provoke tissue injury in diabetes [18]. Hyperglycemia and hypoxia play essential pathophysiological roles in this condition. Knockdown of HIF-1α expression
Conflict of interest
The authors declare no potential conflict of interest.
Acknowledgments
This work was supported by a grant from the Guangdong Science and Technology Program (grant no. 2013B021800324).
References (33)
- et al.
Downregulation of human DAB2IP gene expression mediated by polycomb Ezh2 complex and histone deacetylase in prostate cancer
J Biol Chem
(2005) - et al.
The mechanism of growth-inhibitory effect of DOC-2/DAB2 in prostate cancer. Characterization of a novel GTPase-activating protein associated with N-terminal domain of DOC-2/DAB2
J Biol Chem
(2002) - et al.
Nitric oxide induces hypoxia-inducible factor 1 activation that is dependent on MAPK and phosphatidylinositol 3-kinase signaling
J Biol Chem
(2004) - et al.
Normoxic induction of the hypoxia-inducible factor 1alpha by insulin and interleukin-1beta involves the phosphatidylinositol 3-kinase pathway
FEBS Lett
(2002) - et al.
Insulin resistance, hyperglycemia, and atherosclerosis
Cell Metab
(2011) - et al.
The regulation and activities of the multifunctional serine/threonine kinase Akt/PKB
Exp Cell Res
(1999) - et al.
Adaptive immunity, inflammation, and cardiovascular complications in type 1 and type 2 diabetes mellitus
J Diabetes Res
(2013) - et al.
AIP1 mediates TNF-alpha induced ASK1 activation by facilitating dissociation of ASK1 from its inhibitor 14-3-3
J Clin Invest
(2003) - et al.
Aberrant promoter methylation in human DAB2 interactive protein (hDAB2IP) gene in breast cancer
Clin Cancer Res
(2004) - et al.
Aberrant promoter methylation of human DAB2 interactive protein (hDAB2IP) gene in lung cancers
Int J Cancer
(2005)
AIP1 functions as an endogenous inhibitor of VEGFR2-mediated signaling and inflammatory angiogenesis in mice
J Clin Invest
Intermittent hypoxia is an angiogenic inducer for endothelial cells: role of HIF-1
Angiogenesis
Stabilization of HIF-1alpha is critical to improve wound healing in diabetic mice
Proc Natl Acad Sci U S A.
Synergistic effect of HIF-1alpha gene therapy and HIF-1-activated bone marrow-derived angiogenic cells in a mouse model of limb ischemia
Proc Natl Acad Sci U S A
O2-regulated gene expression: transcriptional control of cardiorespiratory physiology by HIF-1
J Appl Physiol
Functional integrity of nuclear factor kappa B, phosphatidylinositol 3′-kinase, and mitogen-activated protein kinase signaling allows tumor necrosis factor alpha-evoked Bcl-2 expression to provoke internal ribosome entry site-dependent translation of hypoxia-inducible factor 1alpha
Cancer Res
Cited by (10)
Targeting Signalling Pathways in Chronic Wound Healing
2024, International Journal of Molecular SciencesASK1-Interacting Protein 1 Acts as a Novel Predictor of Type 2 Diabetes
2022, Frontiers in EndocrinologyThe involvement of ACO3 protein in diabetic retinopathy through the PI3k/Akt signaling pathway
2022, Advances in Clinical and Experimental Medicine