Hypoxic induction of vascular endothelial growth factor (VEGF) and angiogenesis in muscle by N-terminus Peroxisome Proliferator-Associated Receptor Gamma Coactivator (NT-PGC)-1

both mitochondrial biogenesis and angiogenesis in skeletal muscle. Results : Hypoxic induction of an alternative spliced form of PGC-1alpha induces only angiogenesis in skeletal muscle, and not mitochondrial biogenesis. Conclusion : Alternative splicing of PGC-1alpha explains how PGC-1alpha achieves specific induction of angiogenesis during hypoxia. Significance : The findings suggest novel ways to induce angiogenesis in muscle without simultaneously inducing mitochondrial biogenesis. ABSTRACT The transcriptional coactivator peroxisome proliferator-activator receptor gamma coactivator (PGC)-1 α is required for full hypoxic induction of vascular endothelial growth factor (VEGF) in skeletal muscle cells.


and tube formation, hallmarks of angiogenesis. Transgenic expression of NT-PGC-1α in skeletal muscle in mice induces angiogenesis in vivo. Finally, knockdown of NT-PGC-1α and HIF-1α abrogates the induction of VEGF in response to hypoxia. NT-PGC-1α thus confers angiogenic specificity to the PGC-1α-mediated hypoxic response in skeletal muscle cells.
Skeletal muscle is uniquely adaptable to extracellular physiological cues. For example, endurance exercise triggers mitochondrial biogenesis and neovascularization, ultimately improving fatigue resistance; nerve stimulation controls fiber type composition and maintains progrowth signals; and ischemia triggers complex pathways, including a robust response to hypoxia. The many molecular pathways that regulate these processes are only beginning to be understood.
The transcription coactivator PGC-1α is induced by exercise, nerve stimulation, and hypoxia in skeletal muscle. 1 It is regarded as a crucial regulator of oxidative metabolism in muscle. 2 PGC-1α binds to and augments the activity of several transcription factors at the promoters of nuclear-encoded genes.
Coactivation of nuclear respiratory factor (NRF)-1 and NRF-2 induces the expression of nuclearencoded mitochondrial genes, including almost all genes involved in oxidative phosphorylation (Oxphos). At the same time, PGC-1α indirectly regulates mitochondrial DNA (mtDNA) replication and transcription via increased expression of mitochondrial transcription factor A

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(Tfam) and other nuclear-encoded factors. PGC-1α thus coordinates metabolic gene expression in both the nuclear and mitochondrial genomes. Transgenic expression of PGC-1α in muscle induces functional mitochondrial biogenesis, leading to decreased muscle fatigue and increased running endurance of the mice. 3,4 We recently showed that PGC-1α also dramatically induces angiogenesis, thereby coordinating oxygen/fuel delivery (via vessels) with their consumption (in mitochondria). 5,6 PGC-1α expression is induced by hypoxia in muscle cells, and is required for full hypoxic induction of angiogenic genes like VEGF. PGC-1α induces VEGF by co-activating the transcription factor estrogen-related receptor α (ERRα) on a novel enhancer located in the 1 st intron of the VEGF gene. Transgenic expression of PGC-1α in muscle induces dramatic neovascularization, and these same mice are protected from muscle ischemic injury. 5 Conversely, deletion of PGC-1α in skeletal muscle prevents exercise-mediated angiogenesis. 7 These findings, however, raised a perplexing question.
Hypoxia leads to the induction of VEGF and other hypoxic genes, but does not lead to the induction of mitochondrial genes. If PGC-1α is induced by hypoxia, and if PGC-1α is a potent activator of mitochondrial genes, then why are these genes not induced during hypoxia? We thus hypothesized that a specific form of PGC-1α protein must confer this specificity during hypoxia. Zhang et al. recently reported the existence in liver and brown fat of an alternatively spliced biologically active truncated isoform of PGC-1α, termed NT-PGC-1α. 8 Alternative splicing of the exon6/7 boundary leads to a missence splicing event, the encoding of a premature stop codon, and a protein product less than ½ the size of full length PGC-1α. Ruas et al. demonstrated that transcription of this splice variant is likely initiated from an alternative promoter, and named this alternatively messenger mRNA PGC-1α4. 9 Neither study reported on the effects of these isoforms on angiogenic programs. These shortened PGC-1α proteins retain the sequences necessary for binding to ERRα 10 , but not those required for binding to NRF-1 and NRF-2 11 . We thus reasoned that NT-PGC-1α would likely induce VEGF and angiogenic genes more robustly than mitochondrial genes. We further hypothesized that NT-PGC-1α may be specifically induced by hypoxia, thus conferring specificity to the PGC-1α hypoxic response.

EXPERIMENTAL PROCEDURES
Mice and cells. All animal experiments were performed according to procedures approved by the Beth Israel Deaconess Medical Center Institutional Animal Care and Use Committee (IACUC). Hif-1α floxed mice were from Jackson labs (007568) and PGC-1α KO mice were from Dr. Spiegelman. Isolation and culture of primary skeletal myocytes were performed on entire hindlimb muscle after collagenase/dispase digestion, as described previously. 15 Primary myocytes were differentiated in DMEM (5% horse serum).
Generation of ad-NT-PGC-1α. A bluntend NT-PGC-1α product was generated by PCR from FL-PGC-1α using forward primer consisting of first 32 base-pairs of PGC-1α and reverse primer consisting of 23 base-pairs of homology to the 5' end of exon 6 plus an additional 13 basepairs coding for the additional Leu-Phe-Leu-STOP sequence of NT-PGC1α (For: CACCGGATCCCCGGATCACCACCATGGCT TGG, Rev: GCCTCGAGTTATAAAAACAAAT TTGGTGACTGTGG), and cloned into pAD/CMV/V5-DEST (Gateway).
Real-time PCR. Total RNA was isolated from cultured cells using Turbocapture (Qiagen). Samples for real-time PCR analyses were reverse transcribed (Applied Biosystems), and quantitative real-time PCRs were performed on the cDNAs in the presence of fluorescent dye (SYBR green; Bio-Rad). Relative expression levels were determined using the comparative cycle threshold method. The qPCR data was normalized against control primers for hprt, tbp and 36B4.
Endothelial cell migration assay. Differentiated myotubes in 24-well plates were infected with adenovirus expressing GFP, FL, or NT-PGC-1α for 34 h. BSA or soluble Flt1 (100 ng/ml; R & D Systems) was added to the medium for 12 h. Then, 5X10 4 cells of HUVECs were plated on the upper compartment of transwells (8.0 µm pore size) prewarmed with EBM-2 medium for 16 h at 37°C. HUVEC migration to the lower compartment of transwells was measured after 12 h. Migrated HUVECs were fixed with 4% paraformaldehyde in PBS for 20 min at RT, and cells that remained in the upper compartment were removed with cotton swabs. Cells were blocked with 5% BSA in PBS-Tween 20 (PBST; 0.2% Tween 20) and stained with phalloidin-FITC in PBST for 4 h to visualize filamentous actin. Transwell inserts were washed three times in PBST and mounted onto slides with DAPI mounting medium.
Tube formation assay. HUVEC cells were starved for 16-hours in .5% FBS, EBM-2 media. A 96-well plate was then coated with 50 mcL matrigel per well. 15,000 HUVECs were seeded per well in 100 mcL of starvation media. 1hour post-seeding, the media was removed and replaced with 100 mcL of either NT-PGC-1α or GFP conditioned media. 6 hours later, the HUVECs were examined under a light microscope at 4x magnification. Photographs were taken, and total tube length per field was quantified using ImageJ.
Oxygen Consumption Rate. Primary satellite cells were plated at 2 X 10 5 cells per well and then differentiated into myotubes. Oxygen consumption rates (pmol/min) were assessed using a XF Flux Analyzer (Seahorse Biosciences), at baseline and after the addition of the uncoupler 2,4-dinitrophenol (DNP) to determine maximum respiration rate.
Statistics. Data are presented as means ± standard error of the mean (SEM). Statistical analysis was performed with Student's t-test. P values of less than 0.05 were considered statistically significant.

RESULTS
NT-PGC-1α is preferentially induced by hypoxia in skeletal muscle cells. Oligonucleotides were generated to amplify specifically NT-PGC-1α by quantitative real-time PCR (qPCR) ( Figure 1A). qPCR of plasmids encoding each specific isoform demonstrated the specificity of the qPCR primers ( Figure 1B). To evaluate expression of NT-PGC-1α in skeletal muscle, differentiated primary murine skeletal myotubes were used. We have shown previously that hypoxia induces PGC-1α in these cells 5 , but those results did not evaluate which isoform of PGC-1α was induced. Primary myoblasts were thus made to differentiate in cell culture, and were then treated with 0.5% oxygen for 16 hours, versus normoxic control. Activation of a hypoxic program was confirmed by measuring expression of VEGF, Glut1, and PDK1, genes well-known to be induced by hypoxia ( Figure 1C). As shown in Figure 1D, hypoxia induced the expression of NT-PGC-1α 8-fold. The expression of FL-PGC-1α, on the other hand, was unaltered by hypoxic treatment. Hypoxia thus preferentially induces NT-PGC-1α expression in skeletal muscle cells.
NT-PGC-1α only weakly induces mitochondrial genes in skeletal muscle cells. To evaluate the function of NT-PGC-1α in muscle cells, an adenovirus that expresses NT-PGC-1α was generated. This virus was then used to infect primary myotubes. In order to be sure that any effects are independent of full-length PGC-1α present in myotubes, primary myoblasts were isolated from PGC-1α -/-mice and made to differentiate in culture. Infection of these primary myotubes lacking endogenous PGC-1α led to robust expression of NT-PGC-1α, as measured by Western blotting (Figure 2A). The effects of infection with adeno-NT-PGC-1α were next compared with those of adeno-FL-PGC-1α, versus GFP-only control. Multiplicities of infection of each adenovirus were chosen that achieved equivalent, and moderate, induction of FL and NT-PGC-1α ( Figure 2B). As shown in Figure 2C, FL-PGC-1α led to robust induction of various nuclear genes encoding important components of the mitochondrion, including cytochromes (cycs), electron transport chain complex IV (cox5b), ATPsynthase (Atp5o), the TCA cycle (cs), and mitochondrial replication and transcription (TFAM). In sharp contrast, NT-PGC-1α only marginally induced these same genes ( Figure 2C). Measurements by Western blotting of mitochondrial complex III and V proteins revealed similarly poor induction by NT-PGC-1α ( Figure  2D). NT-PGC-1α thus only weakly induces mitochondrial genes in skeletal myotubes in culture. Finally, to directly and sensitively test the effects of NT-PGC-1α on mitochondrial function, NT-PGC-1α was more strongly overexpressed in primary myotubes, and cellular respiration was measured, using a Seahorse Extracellular Flux Analyzer. As shown in Figure 2E, despite the marked 5-fold greater overexpression of NT-PGC-1α versus FL-PGC-1α in this experiment, oxygen consumption rate (OCR) was only moderately induced by NT-PGC-1α, roughly only half as much as that induced by FL-PGC-1α.

NT-PGC-1α strongly induces VEGF and a pro-angiogenic phenotype in muscle cells.
Infection of PGC-1α -/-myotubes with FL-PGC-1α led to a 3-4 fold induction of the canonical proangiogenic peptide VEGF ( Figure 3A), as we have shown previously 5 . We have shown that the induction of VEGF occurs via coactivation of the nuclear receptor ERRα 5 , and indeed, PGC-1α induces the expression of ERRα itself as well ( Figure 3A). Infection with ad-NT-PGC-1α achieved more than double the induction of VEGF that was seen with FL-PGC-1α ( Figure 3A). This marked induction of VEGF coincided with only minimal induction of mitochondrial genes ( Figure  2C). The ratio of induction of VEGF to cycs by NT-PGC-1α is thus more than 5-fold higher than by FL-PGC-1α ( Figure 3B). NT-PGC-1α directly coactivates ERRα in GAL-luciferase assays Figure  3C), consistent with its ability to induce VEGF 5 and its known ability to bind PPARα 8 .
Endothelial migration and tube formation are hallmarks of angiogenesis. As shown in Figure 3D, conditioned medium from skeletal muscle cells that overexpress FL-PGC-1α led to a 3-fold induction in the migration of endothelial cells in a Transwell assay, as we have shown before 5 . Overexpression of NT-PGC-1α more than quintupled that induction, achieving 15-fold induction of endothelial migration over baseline ( Figure 3D). Addition of soluble fms-like tyrosine kinase 1 (sFlt1), a potent inhibitor of VEGF, completely abrogated the increase in endothelial migration ( Figure 3D), indicating that NT-PGC-1α induces endothelial migration largely via the secretion of VEGF. Lastly, conditioned` medium from myotubes expressing NT-PGC-1α strongly stimulated the formation of tubes by endothelial cells ( Figure 3E). Together, these data indicate that, compared to FL-PGC-1α, NT-PGC-1α strongly favors the activation of a pro-angiogenic program over a pro-mitochondrial program in muscle cells.
NT-PGC-1α induces angiogenesis in vivo. Ruas et al. recently generated transgenic mice that express PGC-1α4 in skeletal muscle, using the muscle creatine kinase (MCK) promoter. PGC-1α4 protein differs from NT-PGC-1α only in its N-terminal 3 amino acids. Both messages contain stop codons in the alternatively spliced exon 7, and thus encode for nearly identical truncated proteins that contain ERR-binding domains but lack NRF-binding domains. We thus used these transgenic mice to test if NT-PGC-1α induces VEGF and angiogenesis in vivo. Tibialis Anterior (TA) muscles were isolated from 12week old animals, and total RNA was prepared, and gene expression measured by qPCR. As shown in Figure 4A, transgenic expression of PGC-1α4 induced total PGC-1α expression approximately 5-fold. Despite this induction, the expression of mitochondrial genes was only marginally induced ( Figure 4B). On the other hand, VEGF expression was significantly induced in these animals ( Figure 4C). PGC-1α4 thus favors the activation of a pro-angiogenic program over a pro-mitochondrial program in intact muscle. Thin frozen sections from the TA muscle were next stained with antibodies against CD31, an endothelial-specific marker that identifies capillaries. As shown in Figure 4B, capillary density, expressed as number of capillaries per high-powered-field, or as capillaries per myofiber, was doubled in transgenic animals. PGC-1α4 thus induces angiogenesis in vivo.
NT-PGC-1α mediates hypoxic induction of VEGF in muscle cells. The transcription factor HIF-1α mediates a large part of the transcriptional response to hypoxia in many, if not most, cells. 12 To separate effects mediated by PGC-1α from those mediated by HIF-1α, we generated cells that lack HIF-1 activity. Primary myoblasts were isolated from animals carrying floxed alleles of HIF-1α. The myoblasts were then infected with adenovirus encoding for the Cre recombinase, leading to inactivation of the HIF-1α locus. The cells were then stably infected with lentivirus expressing shRNA targeted against HIF-1β (ARNT), the obligate heterodimer of both HIF-1 and HIF-2 transcription factors. Finally, the cells were made to differentiate into myotubes. As shown in Figure 5A, HIF-1α and β expression were largely absent in these cells.
Despite strongly reduced HIF-1 expression, however, exposing the cells to 0.2% oxygen still led to significant induction of VEGF ( Figure 5B). Hypoxic induction of VEGF can thus occur HIFindependently in these cells.
We have shown previously that PGC-1α is required for maximal induction of VEGF expression by hypoxia in muscle cells. 5 The data presented here suggest that NT-PGC-1α may be the PGC-1 isoform responsible for this effect. Consistent with this notion, NT-PGC-1α was induced by hypoxia in HIF-minus cells ( Figure  5C) as robustly as in wild type cells (Figure 1). To test the role of NT-PGC-1α directly, lentivirus was generated that encodes for shRNA to target specifically the NT-PGC-1α transcript, while leaving the FL-PGC-1α transcript unaffected. The myoblasts were then made to differentiate into myotubes, and exposed to 0.2% oxygen, versus normoxia control. As shown in Figure 5D, sh-NT-PGC-1α completely abrogated the induction of VEGF in response to hypoxia, while sh-scrambled control had no effect. NT-PGC-1α thus mediates hypoxic induction of VEGF in skeletal muscle cells. The data also indicate that the remnant hypoxic induction was not dependent on lowgrade HIF activity (either HIF-1 or HIF-2) since it was abrogated by shRNA directed at NT-PGC-1α.

DISCUSSION
We show here that NT-PGC-1α, compared to FL-PGC-1α, preferentially induces an angiogenic program over a mitochondrial program in skeletal muscle cells.
PGC-1α induces mitochondrial genes in large part via the coactivation of NRF-1 and NRF-2. The region of PGC-1α that binds to NRF-1 and 2 remains poorly defined, but almost certainly lies outside the peptide sequences retained in NT-PGC-1α. 8,11 On the other hand, NT-PGC-1α retains the LXXLL motifs via which PGC-1α interacts with ERRα, and we show here that NT-PGC-1α can coactivate ERRα. We have shown previously that PGC-1α induces VEGF expression via coactivation of ERRα. 5 We thus propose that the specificity of NT-PGC-1α for the angiogenic program is achieved via specific binding to ERRα, but not NRFs ( Figure 5).
We also show here that NT-PGC-1α is induced by hypoxia in muscle cells, and that NT-PGC-1α mediates hypoxic induction of VEGF. It is important to note that the HIF-1 pathway also contributes to this induction. For this reason, experiments were conducted in the absence of HIF-1 activity (Figure 4). Hypoxic induction of NT-PGC-1α occurs independently of HIF-1 activity, and the induction of VEGF by PGC-1α also occurs independently of HIF-1. FL-PGC-1α has been proposed by others to induce VEGF indirectly via HIF-1, by inducing mitochondrial respiration, leading to elevated consumption of oxygen, local hypoxia, and HIF-1 activation. 13 Our data suggest that this HIF-dependent mechanism is not at play with NT-PGC-1α, because mitochondrial genes are minimally induced. Muscle cells thus appear to activate two entirely separate pathways in response to hypoxia.
In general, prolonged hypoxia tends to repress the expression of mitochondrial complexes in most cell types. Our findings explain why hypoxic induction of PGC-1α does not induce mitochondrial genes, but the findings do not provide a mechanism for the active repression of these genes. The mechanisms remain incompletely understood, and likely differ between cell types. PGC-1α isoforms are not known to repress transcriptional activity, and it thus seems unlikely that they would be involved in this active suppression of Oxphos genes.
NT-PGC-1α largely resides in the cytoplasm. 14 A significant amount of NT, however, must also be nuclear, since NT-PGC-1α robustly coactivates ERRα and induces VEGF expression. It will thus be of interest to evaluate if hypoxia alters cellular localization of NT-PGC-1α. Future studies will also investigate how the expression of NT-PGC-1α is induced by hypoxia. This could occur at a transcriptional level, or during post-transcriptional splicing. We show here that the induction of NT occurs independently of HIF-1 activity, possibly suggesting a posttranscriptional event, such as regulation of alternative splicing.
Cyclic AMP and PKA promote nuclear localization of NT-PGC-1α. 14 We have shown previously that PGC-1α is required for exerciseinduced angiogenesis, and that this occurs in part also via cAMP signaling. 7 Testing if NT-PGC-1α is the principal isoform that mediates exerciseinduced angiogenesis will be of interest, but will likely require specific modification of the murine genome.
Exercise-induced mitochondrial biogenesis, on the other hand, is unlikely to be mediated by NT-PGC-1α, because NT-PGC-1α appears to be a weak inducer of mitochondrial genes in muscle cells, and because PGC-1α appears to be dispensable for exercise-induced mitochondrial biogenesis. 15 Ruas et al. have recently shown that the short forms of PGC-1α regulate an IGF-dependent pro-growth program. It will in the future be of interest to determine comprehensively which gene loci are occupied by NT-PGC-1α, versus FL-PGC-1α, and which genes are unique to NT. It will also be of interest to determine if the pro-growth program remains active under hypoxic conditions.
In summary, the data presented here highlight unique features of NT-PGC-1α in skeletal muscle cells, and explain the paradoxical observation that PGC-1α mediates induction of VEGF, but not mitochondrial genes, in response to hypoxia in these cells.
qPCR amplification of FL-PGC-1α and NT-PGC-1α from FL-PGC-1α and NT-PGC-1α, respectively. C, induction of VEGFA, PDK1, GLUT1 in primary myotubes treated with 0.5% oxygen for 16hrs. D, specific induction of NT-PGC-1α expression by hypoxia in myotubes treated as in C.    A, efficient reduction of expression of HIF-1α and β by combined shHIF-1β expression and adenovirally-mediated Cre expression in primary murine HIF-1α lox/lox myotubes. B, hypoxia induces VEGF and hypoxic genes in primary murine HIF-1α lox/lox myotubes pretreated with shHIF-1β and adeno-Cre, despite severely reduced HIF-1 (as shown in A). C, hypoxia induces NT-PGC-1α despite severely reduced HIF-1, as in B. D, shNT-PGC-1α abrogates the remaining hypoxia response in myotubes with reduced HIF-1 activity treated as in B. *p<.05 by student's t-test. Figure 6. Model of how NT-PGC-1α confers preference for angiogenic genes over mitochondrial genes. FL-PGC-1α binds to ERRα as well as NRFs, allowing for full induction of mitochondrial genes and angiogenic genes. NT-PGC-1α, on the other hand, binds only ERRα, which limits its ability to fully induce mitochondrial genes, while not affecting its ability to induce angiogenic genes like VEGF.

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