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Hypoxia induces an autocrine-paracrine survival pathway via platelet-derived growth factor (PDGF)-B/PDGF-ß receptor/phosphatidylinositol 3-kinase/Akt signaling in RN46A neuronal cells¹

SHELLEY X. L. ZHANG^*, DAVID GOZAL^*,, LEROY R. SACHLEBEN, JR^*, MADHAVI RANE, JON B. KLEIN and EVELYNE GOZAL^*,,2

^* Kosair Children’s Hospital Research Institute, Department of Pediatrics,
Department of Pharmacology and Toxicology,
Kidney Disease Program, Department of Medicine, University of Louisville, Louisville, Kentucky, USA

²Correspondence: KCHRI, University of Louisville, 570 S. Preston St., Louisville, KY 40202, USA. E-mail: Evelyne.gozal{at}louisville.edu

SPECIFIC AIMS

Oxygen deprivation, a frequent occurrence in brain disorders, induces multiple stress-related pathways that regulate proliferation, metabolism, and neuronal survival. The balance between these pathways is critical in determining cellular fate. We examine whether acute hypoxic stimuli will induce an autocrine-paracrine survival response in neurons and assess the role of PDGF-BB and its downstream activation of the PDGF-ß receptor/PI3K/Akt/HIF-1 signaling cascade in this survival pathway.

PRINCIPAL FINDINGS

1. Hypoxia and minimum media growth conditions induce apoptosis in differentiated RN46A neuronal cells with a different time course
Trophic factor withdrawal is a classical stress-related inducer of apoptosis in neuronal cells. To determine whether hypoxia induces apoptosis via a stress pathway comparable to growth factor withdrawal-induced apoptosis, we compared the time courses of RN46A apoptosis induced by hypoxia and by B-27 withdrawal. RN46A cells were cultured in normoxia or exposed to different times of hypoxia (1% O₂) or B-27 withdrawal. Apoptosis was measured using a nucleosomal DNA ELISA kit. No significant apoptosis was detected until 48 h hypoxia, followed by increasing apoptosis, peaking at 96 h (9.1±0.5-fold; P<0.001; n=4). In contrast, after B-27 supplement withdrawal, significant apoptosis was detected starting at 12 h and increasing thereafter (16.3±0.4-fold at 72 h; P<0.001; n=3). The intensity of the apoptotic response to similar durations of B-27 deprivation and hypoxia was significantly higher for B-27-deprived cells starting at 12 h (^#P<0.0001; n=3). These findings suggest that in RN46A neuronal cells, growth factor withdrawal is a more potent inducer of apoptosis than hypoxia.

2. Hypoxia protects RN46A cells from B-27 withdrawal-induced apoptosis
B-27 supplement withdrawal-induced DNA degradation was assessed by DNA laddering (Fig. 1 , lane 3). To examine whether hypoxia induces a survival pathway, cells were exposed to 24 h normoxia or hypoxia in the presence or absence of B-27. Hypoxia significantly attenuated B-27 withdrawal-induced DNA laddering compared with normoxic cells (Fig. 1 , lanes 4 and 3, respectively). These findings indicate that hypoxia induces resistance mechanisms that permit RN46A cells to survive B-27 withdrawal.

View larger version (87K): [in this window] [in a new window]   Figure 1. Hypoxia inhibits growth factors withdrawal-induced cell death. Cells were cultured for 24 h in B-27-supplemented or B-27-depleted media in normoxia or hypoxia (H). Genomic DNA was isolated and DNA laddering was detected by electrophoresis and SYBR gold staining. Hypoxia partially prevented DNA degradation induced by B-27 withdrawal.

3. Hypoxia induces PDGF-B expression in RN46A neuronal cells, and activates PDGF-ß receptor signaling leading to Akt phosphorylation
To determine whether hypoxia induces PDGF-BB production and PDGF-ß receptor activation in RN46A cells, PDGF-B mRNA expression was determined by Taqman real-time quantitative RT-PCR. PDGF-B mRNA levels peaked after 6 h hypoxia and declined to normoxic levels by 24 h. The intracellular expression of PDGF-BB as well as its release into the culture media paralleled the time course of PDGF-BB gene induction. PDGF-ß receptor immunoprecipitates showed that in the absence of PDGF-ß receptor expression changes, PDGF-ß receptor tyrosine phosphorylation increased at 6 h hypoxia, followed by a progressive decrease to normoxic levels by 24 h. Thus, hypoxia induces PDGF-B expression and release in RN46A cells with concomitant PDGF-ß receptor activation. Late reductions in PDGF B expression and PDGF-ß receptor activation at 24 h coincide with the onset of hypoxia-induced apoptosis.

To determine whether hypoxia and PDGF-BB share a common Akt activation pathway, we examined the effect of PI3K inhibition with 25 µM LY294002 on Akt Ser473 phosphorylation in cells exposed to 30 min 30 ng/mL PDGF-BB treatment or 6 h hypoxia. LY294002 abrogated the increase in Akt Ser473 phosphorylation induced by either PDGF-BB or hypoxia. Inhibition of PDGF-ß receptor activation by desensitization of the PDGF-ß receptor with excess PDGF-BB stimulation (400 ng/mL) or by pharmacological inhibition of PDGF-ß receptor tyrosine phosphorylation with10 µM AG1296 significantly decreased hypoxia-induced Akt phosphorylation. These findings implicate PDGF-ß receptor signaling in the activation of Akt by hypoxia and support the concept that initial Akt activation is sustained by increased PDGF-BB release by neuronal RN46A cells during hypoxia.

4. Hypoxia induces a transient phosphorylation of ERK1/2 but a sustained phosphorylation of Akt in RN46A neuronal cells
PDGF signaling cascade involves both ERK/MAP kinase and PI3K/Akt pathways. To examine the contribution of these pathways to hypoxia-induced survival in RN46A cells, we initially compared the time courses of ERK and Akt phosphorylation during hypoxia, as well as after exposure to exogenous recombinant PDGF-BB (30 ng/mL). Both hypoxia and PDGF-BB induced transient ERK activation. In contrast, while PDGF-BB induced early and transient Akt phosphorylation, hypoxia-induced Akt phosphorylation increased at 1 h but remained elevated for up to 24 h of exposure. Neither hypoxia nor PDGF-BB affected total ERK or Akt expression. The divergent time courses of Akt phosphorylation induced by PDGF-BB and hypoxia suggest that sustained release of PDGF-BB ligand during hypoxia underlies the prolonged Akt phosphorylation observed during hypoxia.

5. Hypoxia-induced survival requires PI3K/Akt activation but does not involve ERK activation
To assess the relevance of both ERK and PI3K/Akt pathways to hypoxia induction of survival pathways, apoptosis was induced in RN46A cells by B-27 deprivation and cells were cultured in normoxia or hypoxia for 24 h, in the presence of PDGF-BB (30 ng/mL) with the PI3K inhibitor LY294002 (25 µM) or the MEK/ERK inhibitor PD98059 (10 µM). PDGF BB enhanced cell survival to B-27 withdrawal, as measured by MTT reduction, during normoxic conditions (P<0.05; n=4). In contrast, PI3K inhibition markedly decreased cell viability (P<0.05; n=4) while MEK/ERK inhibition had no effect. Hypoxia increased survival of B-27-deprived cells when compared with normoxic cells. Addition of PDGF-BB or of the MEK/ERK inhibitor did not significantly alter the hypoxia-induced survival response. However, inhibition of PI3K abolished the hypoxia-induced tolerance to B-27 withdrawal (P<0.0001; n=4), suggesting that hypoxia-induced PDGF BB-mediated Akt activation, but not PDGF BB-mediated ERK activation, is critical for cell survival during B-27 deprivation.

6. HIF-1 induction by hypoxia in RN46A cells is mediated by a PDGF-B/PI3K/Akt pathway
Hypoxia inducible transcription factor (HIF-1) is induced by low oxygen levels and translocates to the nucleus to activate and regulate transcription of numerous genes essential to metabolic adaptation, as well as genes that play a role in regulation of cell death (e.g., p53, p21, and Bcl-2). Since recent studies have associated hypoxia induction of HIF-1 transcription factor and HIF-1-dependent gene transcription to the PI3K/Akt pathway, we examined whether the autocrine-paracrine signaling pathway induced by hypoxia in RN46A neuronal cells affected the downstream nuclear binding of HIF-1.

Assessment of HIF-1 DNA binding was carried out by EMSA and by a commercially available ELISA in nuclear extracts. HIF-1 DNA binding activity in RN46A nuclear extracts peaked at 6 h, followed by a decrease at 12 h (Fig. 2 ). PDGF-ß receptor inhibition with AG1296 and PI3K inhibition with LY294002 abrogated the increase in HIF-1 activity (Fig. 2) . These data suggest that in RN46A neuronal cells exposed to hypoxia, activation of PDGF-ß receptor and downstream activation of PI3K/Akt are critical to HIF-1 transcriptional activity.

View larger version (50K): [in this window] [in a new window]   Figure 2. Hypoxia induces HIF-1 DNA binding in RN46A cells in a PDGF/PDGF-ß receptor/PI3K-dependent manner. Cells were pretreated with vehicle (Control), 25 µM LY294002 (LY), or 10 µM AG1296 and exposed to normoxia or hypoxia. Nuclear extract proteins were analyzed for DNA binding activity by A) EMSA or B) using an ELISA method to detect and quantify HIF-1 binding to its consensus sequence. Hypoxia increased HIF-1 binding to DNA after 1 h hypoxia, peaking at 6 h and returning to baseline at 12 h. Inhibition of either PDGF-ß receptor or PI3K abrogated HIF-1 activation.

CONCLUSIONS AND SIGNIFICANCE

Cerebrovascular disorders, pulmonary diseases, or sleep apnea are frequent conditions leading to limited oxygen supply to the central nervous system. All these disorders are accompanied by substantial neurobehavioral morbidity, accounted for by the excessive neuronal cell loss associated with restricted oxygen supply to the neural tissue. Thus, improved understanding of mechanisms underlying adaptation and vulnerability to hypoxia may provide important windows of interventional opportunity to prevent hypoxia-associated neuronal cell loss. We now propose a unique autocrine-paracrine pathway induced by acute hypoxia that involves the release of a growth factor—namely, PDGF BB—which in turn, by binding to its receptor, elicits the activation of an anti-apoptotic pathway involving both PI3K and Akt (Fig. 3 ). Growth factors have been implicated in the prevention or delay of ischemia-induced neuronal apoptosis. Circumstantial evidence based on increased expression of PDGF-BB in the penumbra of ischemic brain infarcts would suggest a similar role for this growth factor. In fact, most neurotrophic peptides will activate upon binding to their specific ligand receptor the Ras/PI3K/Akt signaling pathway, which is critical to neural cell survival. As shown in the present study, inhibition of this pathway leads to abolition of growth factor-induced protection from apoptosis.

View larger version (37K): [in this window] [in a new window]   Figure 3. Novel autocrine-paracrine hypoxia-induced survival pathway in neuronal cells. Treatment of RN46A cells with hypoxia induces PDGF-BB expression and release, with subsequent activation of PDGF-ß receptors. In turn, the activation of PDGF-ß receptor and its tyrosine phosphorylation (PY) activates PI 3-kinase (PI3K), which phosphorylates Akt kinase to activate downstream targets to induce hypoxia-inducible-1 (HIF-1) stabilization and binding to DNA, leading to the enhanced expression and activity of anti-apoptotic pathways.

The downstream survival-related elements recruited by Akt activation are complex. Evidence has been reported on Akt-mediated regulation of the expression or activity of pro- and anti-apoptotic Bcl-2 family members, Akt modulation of pro-and anti-apoptotic gene transcription via HIF-1, Forkhead, NF-B, or CREB transcription factors or by Akt direct interference with cellular metabolism. We identify PDGF-ß receptor-dependent activation of PI3K/Akt signaling pathway as a critical pathway underlying downstream HIF-1-mediated gene transcription in RN46A neuronal cells.

In summary, neuronal cells exposed to severe hypoxia display a biphasic survival response characterized by increased cellular protection, followed by programmed cell death. In the early phase of the hypoxic response, the increased expression and release of PDGF-BB will permit cell survival to any proapoptotic stimulus by activating the PDGF-ß receptor/PI3K/Akt pathway. Thus, selective targeting of this pathway may provide unique opportunities to preserve neuronal cell survival in many of the pathological conditions involving hypoxia-induced cell death.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-1111fje; doi: 10.1096/fj.02-1111fje

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