Hypoxia Induces Expression and Activation of AMPK in Rat Dental Pulp Cells

Abstract

Rat dental pulp RPC-C2A cells, established and kindly provided by Kasugai et al. (1988), were maintained in minimum essential medium (MEM, Invitrogen, Carlsbad, CA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 10 mg/mL kanamycin at 37°C in a humidified atmosphere at 5% CO 2 with 20% O 2 . As hypoxic conditions, cells were cultured in a humidified atmosphere at 5% CO 2 with 2% O 2 . Cell Proliferation AssayCells were seeded in 96-well plates at a density of 5 x 10 4 cells/well and cultured for 24 hrs, and then further cultured for an additional 6, 12, or 24 hrs under normoxic or hypoxic conditions. Cell viability was measured by water-soluble tetrazolium salt (WST-1) reduction activity, with a cell counting kit (Dojindo, Kumamoto, Japan). WST-1 was added at a final concentration of 0.5 mM and ABSTRACT AMP-activated protein kinase (AMPK) is a stressresponsive enzyme involved in cell adaptation to an energy crisis. We hypothesized that hypoxia suppresses oxidative phosphorylation and ATP production, resulting in AMPK activation to protect cells. We investigated the effects of hypoxia on cell proliferation, the expression of AMPK and hypoxia-inducible factor 1␣ (HIF-1␣), the activation of AMPK, and the relationship between AMPK and HIF-1␣ expression in rat dental pulp RPC-C2A cells. AMPK in the cells was composed of catalytic ␣1, and regulatory ␤1 and ␥1 subunit isoforms. Cell proliferation was initially suppressed under hypoxia, but it increased thereafter, together with an increase in the expression of AMPK and HIF-1␣, and the activation of AMPK. Down-regulation of AMPK␣1 by siRNA inhibited cell proliferation under both normoxia and hypoxia, revealing that AMPK induction and activation were required for cell proliferation, although HIF-1␣ expression under hypoxia was not affected.

Hypoxia Promotes Mineralization of Human Dental Pulp Cells

“…The response of DPCs to hypoxia is a complex biological process that contains multiple aspects, including the change of cell basic activity as well as the appropriate inductive molecular signals. The underlying cellular and molecular mechanisms regulating the events reacting to hypoxia have been the targets of a series of investigations (2)(3)(4)(5)(6)(7). Although these studies have provided valuable information about the effects of hypoxia on DPC cell cycle, energy, proliferation, stemness, angiogenic ability, and erythropoietin expression, little information was available regarding DPC mineralization in hypoxia.…”

“…It has been reported that short-term hypoxia had effects on the growth of DPCs to induce cell cycle arrest and apoptosis (2). DPCs changed cellular energy status to adapt to hypoxia environment by activating adenosine monophosphate-activated protein kinase (3). However, a long-term hypoxia condition promoted dental pulp stem cell proliferation and maintained their stemness (4).…”

Effect of Proinflammatory Cytokines on the Expression and Regulation of Human Beta-Defensin 2 in Human Dental Pulp Cells

“…Recent studies have suggested that AMPK plays an important role in the treatment of type 2 diabetes mellitus, protection from apoptosis, and regulation of angiogenesis; exerts antiatherosclerotic effects; and is involved in anti-inflammatory signaling (17,18). Furthermore, the levels of the main AMPK subunit isoform increased time dependently under hypoxia in rat dental pulp cells (19). This suggests that AMPK plays an important role in the reactions of pulp cell to stimuli, presumably in maintaining energy homeostasis.…”

Targeting AMPK for Therapeutic Intervention in Type 2 Diabetes

“…On the other hand, TNF alters AMPK activation by modulating the synthesis of PP2C (Lu et al, 2010;Steinberg et al, 2006). Aside from the rapid control of AMPK activation by phosphorylation, changes in the expression of subunit genes or the turnover of AMPK subunits can help to fine tune AMPK activity in some tissues (Barry et al, 2010;Fukuyama et al, 2007;Hallows et al, 2006;Qi et al, 2008;Niesler et al, 2007;Steinberg et al, 2003).…”