Hypoxia induces voltage-gated K + ( Kv ) channel expression in pulmonary arterial smooth muscle cells through hypoxia-inducible factor-1 ( HIF-1 )

Hypoxia-inducible factor- (HIF-) regulates the expression of hypoxia-inducible genes by binding erythropoietin (EPO) enhancer fragments. Of these genes, HIF- upregulates voltage-gated K+. channels (Kv.) in rat PC cells. Whether HIF- regulates hypoxia-induced Kv channel expression in cultured pulmonary artery smooth muscle cells (PASMCs), however, has not been determined. In this study, we investigated the eff ects of hypoxia on the expression of Kv. Kv., Kv., and Kv. channels in PASMCs and examined the direct role of HIF- by transfecting either wild type or mutant EPO enhancer fragments. Our results showed that  h exposure to hypoxia signifi cantly increased the expression of Kv., Kv., Kv., and Kv.; and this hypoxiainduced upregulation was completely inhibited after transfection with the wild type but not mutant EPO enhancer fragment. Th ese results indicate that HIF- regulates hypoxia-stimulated induction of Kv. Kv., Kv., and Kv. channels in cultured PASMCs. ©  Association of Basic Medical Sciences of FBIH. All rights reserved


INTRODUCTION
Effi cient gas exchange in the lungs is maintained through a mechanism known as hypoxic pulmonary vasoconstriction (HPV), which serves to match local perfusion to ventilation and optimize arterial blood gas exchange.Hypoxia has recently been shown to induce pulmonary artery contraction, even after the endothelium has been denuded [, ].Th is hypoxia-induced constriction has been demonstrated to occur by inhibition of voltage-gated potassium channels (Kv) at the single pulmonary artery smooth muscle cell level.Voltage-gated potassium channels serve to depolarize the membrane potential, which in turn opens voltage-gated calcium channels.Th e rise in cytoplasmic calcium concentration within the cell activates the contractile apparatus.Previous studies have shown that Kv channels serve an important role during HPV [, -].At the molecular level, Kv channels are composed of pore-forming α-subunits and the cytoplasmic regulatory (auxiliary) β-subunits [].To date, nine families of Kv channels α-subunits have been identifi ed, and each of these subunit groups have multiple subtypes.Based on patch-clamp recording studies using blocking antibodies, four candidate Kv channel α-subunits which could form O-sensitive channels have been identified: namely, Kv., Kv., Kv., and Kv. [-].Following in vitro or in vivo exposure to chronic hypoxia of more than  h, Kv., Kv., Kv., and Kv. channel gene and protein expression were all reduced.Hong et al recently found that even shorter hypoxia durations of  to  h in vivo resulted in decreased gene transcription of these four Kv channels [].However, there have been few studies on how shorter hypoxia exposures alter the mRNA and protein expression of these particular Kv α subunits (Kv.,Kv., Kv. and Kv.) in cultured rat PASMCs.Furthermore, the molecular mechanisms underlying HPV have not been fully elucidated.Hypoxia-inducible factor- (HIF-) is a key mediator of pulmonary response to hypoxia [].HIF- is a heterodimeric transcription factor consisting of HIF-α and HIF-β subunits.Although HIF-β is constitutively expressed in the lung, expression of HIF-α is tightly regulated by O tension.Exposure of PASMC to hypoxia signifi cantly increases HIF-α expression [].HIF-α translocates to the nucleus, dimerizes with HIF-β, and binds to hypoxia response elements, which contain the erythropoietin (EPO) gene enhancer [, ].HIF- recognizes an  bp DNA motif ' TACGTGCT-' in the EPO enhancer, but not to a mutated sequence containing a ' nucleotide substitution that eliminates the enhancer function [, ].Hypoxia signal transduction depends to some extent on hypoxia-activated HIF- binding to the EPO enhancer or a similar sequence [, ].Thus, we hypothesized that HIF- may be involved in the hypoxia-induced oxygen-sensitive Kv channel expression.The aim of the current research was to investigate the effects of hypoxia on the gene and protein expression of Kv., Kv., Kv., Kv. channels in rat PASMCs, using RT-PCR and immunoblotting.Transient transfection with either wild or mutant EPO'-enhancer fragments was used to determine the role of HIF- and reveal HPV-dependent molecular mechanisms.

MATERIALS AND METHODS
Cell culture and hypoxia exposure.Primary cultured PASMCs were prepared from  male Sprague-Dawley rats (- g) as described previously [].Our animal protocol was approved by the Institutional Animal Care and Use Committee of the Huazhong University of Science and Technology, Tongji Medical School and conforms to current guidelines from the National Institutes of Health and the American Physiological Society for the use and care of laboratory animals.Briefl y, the rats were anesthetized with urethane, and the heart and the lungs were removed rapidly and immediately placed in ice-cold D-Hanks solution.The intrapulmonary arteries ( th - th divisions) were dissected free of adventitia and the endothelium was removed with a cotton swab.Th e remaining smooth muscle was first digested in  mg/ml collagenase (Worthington Biochemical Company) for  to . hours and then in . mg/ml trypsin (Worthington Biochemical Company) for  min at °C.DMEM/ fetal bovine serum (FBS) was added to the solution to stop digestion.Cells were incubated in a humidified atmosphere of  CO in air at °C for  to  days and fed twice weekly with DMEM/ FBS supplemented with  U/ml penicillin, and  μg/ml streptomycin (BioFluids, Camarillo, CA).Th e primary cultured PASMCs were subcultured at a : ratio.Passages two to four were used in this study.Twenty four hours before the experiments, the media was replaced with DMEM-.FBS supplemented with  U/ml penicillin, and  μg/ml streptomycin in order to stop cell proliferation.The purity of the PASMCs in the primary cultures was confirmed by immunocytochemistry using a specific monoclonal antibody raised against smooth muscle α-actin.All the cells reacted with this antibody, in-dicating that the cultures contained only PASMCs.For hypoxic incubations, plates were transferred to an oxygen-regulated incubator containing a mixture of . N,  CO, and . O (partial pressure of oxygen .-.kPa) for  h.Control (normoxic) plates were cultured in  N,  CO, and  O.Th ere were no signifi cant changes in pH values of the culture media or in cell viability following  h incubation under either normoxic or hypoxic conditions.Transfection.PASMCs were transiently transfected with Epo enhancer fragments cloned into pRL-SV by Lipofectamine method according to manufacturer's protocol(Gibco BRL, Gaithersburg, MD).The EPO enhancer fragments were designed as follows: wild fragments (W) were: '-agcttGCCCTACGTGCTGTCTCAg-' (forward) and '-aCGGGATGCACGACAGAGTCttaa-' (reverse), and the mutant fragments (M) were: '-agcttGCCCTAAAAGCTGTCTCAg-' (forward) and '-aCGGGATTTTCGACAGAGTCttaa-' (reverse).Preparation of total RNA.Total RNA was obtained from quiescent passage four PASMCs using TRIzol Reagent according to the manufacturer's instructions.Isolated total RNA was dissolved in RNase-free water at μg/μl and stored at -°C.Semiquantitative reverse transcription polymerase chain reaction.RT-PCR was performed with TaKaRa RNA PCR Kit Ver.. according to manufacturer's instructions (TaKaRa Biotechnology, Co., Ltd. Dalian, China).Th e sense and antisense PCR oligonuleotide primers (Table ) for cDNA amplification were specifically designed from coding regions of specifi c Kv channels as described previously [].

Statistical analysis
The data were expressed as mean±SEM.Intergroup comparisons are made using a factorial ANOVA with post hoc testing using the Fisher least significant differences test.A p<. was considered statistically significant.

RESULTS
Total RNA was extracted from passage - rat PASMCs incubated under normoxia or hypoxia for  h.Gene transcription (mRNA levels) of Kv channel α subunits (Kv.,Kv., Kv., and Kv.) were examined, and the β-actin mRNA level was used as control.
The mRNA levels of Kv., Kv., Kv., and Kv. were significantly increased after exposure to hypoxia for  h (Figure , p<. for all).To understand the role of HIF- in hypoxia induced stimulation of K + channel gene expression, we examined Kv.

DISCUSSION
The goals of this study were to evaluate the effects of hypoxia on Kv α subunit expression in PASMCs and to determine the role of the EPO enhancer element on Kv α subunit gene and protein levels.The major findings of this study were that: (a)  h hypoxia exposure increased the expression of PASMC Kv α subunits (Kv.,Kv., Kv., and Kv.) at the transcriptional level; (b) protein expression of Kv., Kv., and Kv. channels were significantly increased after  h hypoxia and (c) the eff ects of  h hypoxia were completely blocked following transfection with wild type but not mutant EPO-enhancer fragment.Our results suggest that HIF- regulates hypoxia-induced expression of Kv. Kv., Kv., and Kv. channels in cultured PASMCs through EPO enhancer effects.Th e activity of certain Kv channel subtypes has been shown to be O sensitive.Kv channels are important determinants of vascular tone control, and the role of Kv channels have been investigated in several models of chronic hypoxia.Shortly after the fi rst report on the effect of acute hypoxia on K + channels was published [], Smirnov et al. [] demonstrated that PASMCs of adult rats subjected to four weeks in an hypoxic environment have reduced Kv current compared with normoxic rats.It had been proposed that the observed reduction in Kv current amplitude is a result of decreased channel expression.Th e fi rst evidence for down-regulation of specifi c Kv channel α-subunits (Kv. and Kv.) in cultured rat PASMCs under chronic hypoxia (- h) was provided by Wang et al [].More recently, it was reported that chronic hypoxia also decreases the mRNA expression of Kv., Kv., Kv., Kv., and Kv. α subunits in cultured rat PASMCs [, ].Likewise, PASMCs isolated from chronically hypoxic animals also showed reduced levels of these particular channels [-].However, there are no previous reports of the eff ects of  h hypoxia on mRNA and protein expression of Kv α subunits (Kv.,Kv., Kv., and Kv.) in cultured rat PASMCs.In contrast to the results for the longer exposure times, we found that the expression of Kv., Kv., Kv., and Kv. channels increased at  h of hypoxia stimulation.A similar observation has been reported by Conforti and Millhorn [], who demonstrated that gene expression of Kv. in cultured PC cells was signifi cantly upregulated after  h exposure to hypoxia.Interestingly, Hong et al. [] found that mRNA expression of Kv., Kv., and Kv. were all signifi cantly downregulated in freshly isolated rat PASMCs that had previously been exposed to hypobaric hypoxia for  h.Th is diff erence in expression levels could be attributed to diff erences in in vivo and in vitro exposure to hypoxia, to diff erences between cultured and freshly isolated cells, and/ or to diff erences between  and  h of exposure.Upregulation of Kv., Kv., Kv., and Kv. channel expression by  h hypoxia suggests an adaptive response following functional inhibition of Kv in response to acute hypoxia.HIF- is a heterodimeric transcription factor responsible for activation of many hypoxia-inducible genes including EPO and endothelin- [, , ].To date, most of the hypoxiainducible gene expression systems have focused on the hypoxia responsive promoters/enhancer, which harbor HIF- binding sites.EPO enhancer fragments are located in the '-fl anking region, and in combination with HIF-, promote gene transcription [, ].Since the mechanism underlying hypoxic induction of gene expression via the HIF- pathway has been well characterized, we hypothesized that hypoxia induced Kv., Kv., Kv., and Kv. channel gene upregulation could occur through HIF-.To test our hypothesis, we transfected external EPO-enhancer fragments (wild-type or mutant) to test the eff ect of the enhancer on Kv channel expression.As reported in a previous study, the external EPO-enhancer fragments can compete with internal EPO-enhancer fragments (or similar sequences) to bind with HIF- in the cytoplasm and prevent HIF- entry into the nucleus, thereby inhibiting gene transcription [].Th e mutant EPO-enhancer fragments, with a "CGT" to "AAA" substitution in its sequence, eliminates HIF- binding as demonstrated by Semenza and colleagues [, , ].Th e current study found that the upregulation of Kv., Kv., Kv., and Kv. channel expression after exposure to  h hypoxia was completely blocked by transfection with the wild type of EPO-enhancer fragment, but their expression was unaff ected by transfection with the mutant EPOenhancer fragment.Th ese results are in agreement with a previous study, which showed that expression of HIF- is required for hypoxia-induced alterations in the Kv current [].In a conclusion, our results demonstrate that exposure to  h of hypoxia upregulated the expression of Kv., Kv., Kv., and Kv. channels in PASMCs, and this increase in gene expression is regulated through a HIF--dependent mechanism.

DECLARATION OF INTEREST
Th e authors declare no confl ict of interest.

TABLE 1 .
Oligonucleotide sequences of the primers used for RT-PCRThe cDNA samples were amplified by denaturing at °C for  min, annealing at -°C for  min, and extending at °C from  min.After  cycles, the PCR products were electrophoresed through a . agarose gel, and amplified cDNA bands were visualized by ethidium bromide staining.
All the sense and antisense primers were synthesized by the Shanghai Biological Company.
PASMCs incubated under normoxia or hypoxia.Since the Kv. antibody was not available, only Kv., Kv., and Kv. protein levels were examined.The α-actin protein level was used as control.
ment completely blocked the hypoxia-induced increase in Kv., Kv., Kv., and Kv. mRNA expression.In contrast, transfection with the mutant EPO-enhancer fragment did not affect hypoxia-induced increase in Kv. Kv., Kv., and Kv. mRNA expression (Figure).Immunoblotting was used to compare protein levels of the channels in