Complex role of protein phosphorylation in gene activation by hypoxia

Kidney International, Vol. 51(1997), pp. 556—559

Complex role of protein phosphorylation in gene activation by hypoxia SUSANA SALCEDA, INGRID BECK, VICKRAM SRINIVAS, and JAIME CARO Cardeza Foundation for Hematologic Research, Thomas Jefferson University, Philadelphia, Penmylvania, USA

Complex role of protein phosphorylation in gene activation by response to hypoxia nor expressed hypoxia responsive genes. The hypoxia. Mammalian cells are able to sense decreased oxygen studies in ARNT deficient cells also showed that HIF-la and 13 tension in their environment and turn-on the expression of mRNAs levels did not change under hypoxic conditions, indicatspecific hypoxia responsive genes. The best studied of these ing that HIF-1 complex formation was not regulated at the mRNA hypoxia regulated genes is the one that encodes for erythropoie- level of its protein components. The unresponsiveness of HIF-1 a tin, the glycoproteiri hormone that regulates red cell production and /3 mRNA to hypoxia was confirmed in Hep3B cells. As shown [1]. The response of the erythropoietin gene to hypoxia is by the Northern blot in Figure 1, exposure of Hep3B cells to mediated by an enhancer sequence located at the 3' flanking hypoxia (Hx), cobalt (Co) or desferrioxamine (Dfx) from two to region of the gene [2—4]. A hypoxia inducible DNA-binding sixteen hours show no change in either ARNT or HIF-la protein complex, termed HIF-1, regulates the transcriptional mRNAs, whereas the mRNA for erythropoietin accumulates in function of the erythropoietin enhancer [5]. Most interestingly, time. These results clearly indicate that HIF-la protein is reguidentical HIF-1 complexes also control the responses of other lated either at the level of its translation or by modifications of its hypoxia regulated genes, in what appears to be a general mecha- rate of degradation. Since ARNT protein is constitutively exnism of oxygen sensing and response [6, 7]. These other hypoxia pressed, HIF-1 complex DNA-binding activity is likely controlled regulated genes include vascular endothelial growth factor, glyco- by the availability of the HIF-la subunit. lytic enzymes such as pyruvate kinase and aldolase A, glucose Protein phosphorylation and signal transduction transporter I and endothelin, among others. All these genes are transcriptionally activated by hypoxia and also by transition metals The mechanisms of oxygen sensing and signal transduction in such as cobalt (Co) and by iron chelators such as desferrioxamine the hypoxia response are still unknown. Protein phosphorylation (Dfx) [reviewed in 8]. is a widely used mechanism of signal transduction that has also Recently Wang and Semenza purified the protein components been implicated in gene activation by low oxygen tension [14—16]. of the HIF-1 DNA-binding complex [9, 10]. Their biochemical To study the role of phosphorylation in the response to hypoxia purification revealed the presence of one subunit of about 120 we utilized several protein kinase inhibitors of different specificity. kDa (HIF-la) and a second subunit, HIF-1f3 with polypeptides of These studies were initially conducted in B-I cells, a permanently 91, 93 and 94 kDa with a similar tryptic digestion composition. transfected Hep3B derived cell line expressing a Luciferase Cloning of the corresponding cDNAs showed that both subunits reporter eDNA under the control of a minimal erythropoietin belong to a subfamily of basic-helix-loop-helix (b-HLH) transcrip- promoter and the hypoxia responsive enhancer. These cells give a tion factors containing a PAS domain. HIF-Ia resulted to be a very sensitive and reproducible Luciferase response to hypoxia, newly recognized member of the group while HIF-1/3 turn out to cobalt and desferrioxamine. Results with the use of H-7, a rather be the already described aryl hydrocarbon receptor nuclear general protein kiriase inhibitor, showed an almost complete translocator (ARNT) protein. These two proteins appear to form inhibition of the Luciferase response to all three stimuli as a heterodimer complex which then interact with the putative depicted in Figure 2A. These initial results confirmed the role of hypoxia-enhancer sequences. The mechanisms involved in the protein phosphorylation in the hypoxia response. No effect on hypoxic induction of this DNA-binding complex are still not clear. Luciferase expression was observed with the use of Wortmannin, a P1-3 kinase inhibitor (not shown), whereas Calphostin C, a Regulation of HIF-1 complex formation protein kinase C inhibitor, was a strong inhibitor against Co and ARNT is also the partner of the aryl hydrocarbon receptor Dfx, but showed poor activity against hypoxia (Fig. 2B). Genistein, (AhR) where in the heterodimeric complex regulates the xenobi- a protein tyrosine kinase inhibitor was also a potent inhibitor otic response of several genes including those of the cytochrome against all three stimuli as shown in Figure 3A. The inhibition P450 family [11]. The absolute requirement of ARNT in the observed by these compounds on Luciferase activity was further hypoxia response was recently demonstrated in ARNT deficient evaluated by Northern blot analysis of the endogenous erythrocells [12, 131. These cells neither induced HIF-1 binding activity in poietin gene (Fig. 3B), which confirmed the absolute inhibitory effect of Genistein (G) and the partial effect of Calphostin C (C). © 1997 by the International Society of Nephrology

Also shown in this Figure is the lack of effect of these inhibitors on the expression of HJF-1 a or /3 mRNAs.

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The marked inhibitory effect of Genistein, as also reported originally by Wang et al [161, prompted us to use PD 098059, a selective MEK inhibitor [17]. MEK1 and MEK2 are dual-speci-

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mediated responses [18]. As shown in Figure 4A, PD 098059 produced a dose dependent inhibition of the Luciferase response

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study the mechanism of action of PD 098059, we evaluated its effects on HIF-1 complex formation. Nuclear extracts obtained from normal, 1-Ix, Co and Dfx treated cells were analyzed using gel shift assays with a radiolabeled HIF-1 binding site. Surprisingly, as

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Fig. 2. Effect of protein kinase inhibitors on Luciferase activity in B-i cells.

A. Cells were exposed to hypoxia (LI Fix), cobalt (+ Co) or desferrioxshown in Figure 4B, PD 098059 had no effect on HIF-1 DNA amine (K Dfx) for eight hours in the presence of increasing concentrabinding activity induced by either stimulus. These results suggest tions of H-7. Symbol • is normal. B. Cells were exposed, as above, to first that there could be dissociation between DNA-binding increasing concentrations of Calphostin C.

activity and transcriptional activation, and second, it indicates that protein phosphorylation is likely involved in the trans-activating

function of the HIF-1 complex. This lack of effect of protein The transmission of these signal is usually mediated by a network phosphorylation on DNA-binding activity was further confirmed of interacting protein kinases that governs a large number of by the treatment of nuclear extracts with protein phosphatases, cellular processes, including the transcriptional activation of which showed no effect on DNA-binding activity (not shown). A similar dissociation between DNA-binding and transcriptional activation was recently described for the stress-induced phosphorylation and activation of the transcription factor CHOP, a member of the C/EBP family [191. Although significant advances have been made recently in our understanding of the mechanisms that regulate the responses to hypoxia, major questions still remain unanswered. Within these unknowns are the signal transduction pathways that determine the formation of the HIF-1 complex and the mechanisms used by this complex to increase transcriptional activity. Protein phosphoiyla-

genes. The studies presented here clearly confirm early reports of the involvement of protein phosphorylation events in the signal transduction of the hypoxia response. The pattern of phosphorylation is, however, rather complex. Inhibitors of protein kinase C were very effective in blocking the response to cobalt and desferrioxamine, but had little effect in the response to hypoxia. The tyrosine phosphorylation inhibitor Genistein was very effective against all stimuli and blocked both HIF-1 binding activity and gene expression. On the other hand, PD 098059, a highly specific inhibitor of the MAP pathway, inhibited gene expression, but not HIF-1 DNA-binding (Note added in proof). The results presented

tion is a common mechanism utilized by the cell for the rapid have just outlined the complexities of the signal tranduction transmission of extracellular signals to their intracellular targets. pathways that culminate in gene activation in response to hypoxia.

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Salceda et a!: Gene activation by hypoxia

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Fig. 3. A. Effect of Genistein on Luciferase expression by B-I cells. Cells were exposed, as in Figure 2, to Genistein for eight hours. Symbols are: cobalt. B. Northern (U) normal; (LII) hypoxia; (•) desferrioxamine; blot analysis of total RNA obtained from normal (N) or stimulated B-i

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Fig. 4. Effect of PD 098059 on the response to Hx, Co and Dfx by B-i cells. A. Cells were exposed for 6 hours to the different agents, as above, in the presence of increasing concentrations of PD 098059. Symbols are: (U) N; Co. B. Gel shift assay utilizing nuclear extracts from (LI) Hx; (•) Dfx; normal and treated cells, as above, in the absence (—) or presence (+) of 50 ILM of PD098059. Labeled probe from the Epo 3' enhancer.

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More studies will be needed to determine the mechanism by which low oxygen tension initiates these events, the nature of the protein kinases involved and the exact role of phosphorylation in the transactivation activity of the HIF-l complex.

References Acknowledgments This work was supported by an NIH grant DK-34642, and by grants from the Juvenile Diabetes Foundation International #195009 and the Southeastern Pennsylvania Affiliate of the American Heart Association. Susana Salceda is supported by an Individual Training Grant from the N.H.L.B. Institute #1 F32 HL09247. Reprint requests to Dr. Susana Salceda, Cardeza Foundation fbr Hematologic Research, Thomas Jefferson University, 1015 Walnut St., Philadelphia, Pennsylvania 19107, USA.

Note added in proof Although PD 09859 has been described as a specific inhibitor of MEK [171, preliminary results from our laboratory suggest that it could also affect the activity of P450 enzymes. The possible confounding effects of this finding on our results are currently being investigated.

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