STAT3 precedes HIF1α transcriptional responses to oxygen and oxygen and glucose deprivation in human brain pericytes

Abstract

Brain pericytes are important to maintain vascular integrity of the neurovascular unit under both physiological and ischemic conditions. Ischemic stroke is known to induce an inflammatory and hypoxic response due to the lack of oxygen and glucose in the brain tissue. How this early response to ischemia is molecularly regulated in pericytes is largely unknown and may be of importance for future therapeutic targets. Here we evaluate the transcriptional responses in in vitro cultured human brain pericytes after oxygen and/or glucose deprivation. Hypoxia has been widely known to stabilise the transcription factor hypoxia inducible factor 1-alpha (HIF1α) and mediate the induction of hypoxic transcriptional programs after ischemia. However, we find that the transcription factors Jun Proto-Oncogene (c-JUN), Nuclear Factor Of Kappa Light Polypeptide Gene Enhancer In B-Cells (NFκB) and signal transducer and activator of transcription 3 (STAT3) bind genes regulated after 2hours (hs) of omitted glucose and oxygen before HIF1α. Potent HIF1α responses require 6hs of hypoxia to substantiate transcriptional regulation comparable to either c-JUN or STAT3. Phosphorylated STAT3 protein is at its highest after 5 min of oxygen and glucose (OGD) deprivation, whereas maximum HIF1α stabilisation requires 120 min. We show that STAT3 regulates angiogenic and metabolic pathways before HIF1α, suggesting that HIF1α is not the initiating trans-acting factor in the response of pericytes to ischemia.

Fig 1. OGD produces a stronger gene regulatory response than hypoxia and glucose deprivation alone.

(A) Normalised log2 transformed gene expression data was compared in a box plot to ensure that normalisation was equal between the samples of the bead-array. Error bars show standard deviation (SD). (B) PCA-plot of the top 500 probe sets p<0.05 comparisons to control to show that the replicates within the gene expression data group according to treatments. (C, E) Venn diagrams were constructed from the upregulated genes, (C) fold change (fc) >1.5 with a p-value <0.05 after 2hs or (E) 6 hs of: Control, Hypoxia, or oxygen and glucose deprivation (OGD). (D, F) Venn diagrams of down regulated genes fc<0.66, p-value <0.05 in (D) 2h or (F) 6 h treatments as in (C, E).

Fig 2. HIF1α is not the primary early transcription factor regulating gene expression in hypoxic or OGD–treated pericytes.

ChIP-seq data for c-JUN, NFκB, STAT3 or HIF1α were analysed for binding peaks near (5Kbp) the transcriptional binding site of genes regulated (fc>1.5, p<0.05) in hypoxic or OGD treated pericytes. Venn diagrams are for (A) 2h hypoxia, (B) 6h hypoxia, (C) 2h OGD or (D) 6h OGD-treated pericytes. The numbers of genes regulated by either of the four transcription factors (x) compared to the total number of regulated genes (y) are presented as (x/y) below each graph.

Fig 3. STAT3 drives both hypoxic and OGD responses stronger than HIF1α in 2h treated pericytes.

Bound regulated genes (BRGs) with transcription factor occupancy within 5Kbp of a TSS for STAT3 and HIF1α were identified in by comparing ChIP-seq data to the pericytic Illumina gene expression data. (A) HIF1α alpha BRGs in the 2h hypoxic condition (fc>1.5, p<0.05) was plotted in a heat map. Panel (B) shows HIF1α BRGs in the OGD condition plotted in a heat map. (C) STAT3 BRGs after 2h of hypoxia and (D) 2h OGD. (E) A Venn diagram was constructed to highlight the differences in STAT3 or HIF1α BRG distribution between 2h hypoxic or OGD conditions.

Fig 4. STAT3 dominates the OGD responses but shows equal activity to HIF1α in the hypoxic responses in 6h treated pericytes.

STAT3 or HIF1α bound regulated genes (BRGs) with binding site(s) 5Kbp of a TSS were identified by comparing ChIP-seq data to the pericytic Illumina gene expression data. (A) HIF1α alpha BRGs in the 6h hypoxia or (B) OGD condition (fc>1.5, p <0.05) was plotted in a heat map. Panel (C) shows STAT3 BRGs in the hypoxia and (D) OGD condition plotted in a heat map. (E) A Venn diagram was constructed to highlight the differences in STAT3 or HIF1α BRG distribution between 6 h hypoxic and OGD conditions.

Fig 5. STAT3 BRGs c-MYC and GDF are upregulated 2h after treatment.

QPCR of normoxic, hypoxia or OGD treated SVZ pericytes with 2h or 6h treatments. SVZ pericytes were analysed for differences in expression of the indicated transcripts with qPCR (n = 3) and compared to Illumina data (n = 4 or n = 3). The individual analysed transcripts were grouped into non-bound (not bound by either STAT3 or HIF1α as BRGs), STAT3 BRGs, HIF1α/STAT3 combined BRGs or HIF1α BRGs. Statistic analysis was made using 2-way ANOVA and error bars represent standard deviations of the mean (SEM); *p<0.05, ** P<0.01, ***p<0.001, ****p<0.0001.

Fig 6. PSTAT3 is increased in the nucleus of pericytes after 5min of OGD or hypoxia.

Localization of pSTAT3 and HIF1a over time. (A) Confocal images showing pS727STAT3 (green), DAPI (blue) and Phalloidin (red) after 5 minute treatment of Normoxia (upper panel), OGD (middle panel) and OGD + Glucose (lower panel). (B) Confocal images showing pS727STAT3 (green), DAPI (blue) and Phalloidin (red) after 120 minute treatment of normoxia (upper panel), OGD (middle panel) and hypoxia (lower panel). (C) Confocal images showing HIF1α (green), DAPI (blue) and Phalloidin (red) after 5 minute treatment of Normoxia (upper panel), OGD (middle panel) and hypoxia (lower panel). (D) Confocal images showing HIF1α (green), DAPI (blue) and Phalloidin (red) after 120 minute treatment of Normoxia (upper panel), OGD (middle panel) and hypoxia (lower panel). (E) Quantification of nuclear intensity of pS727STAT3 in all conditions at 5 and 120 min. (F) Quantification of nuclear intensity of pS727STAT3 in all conditions at 5 and 120 min.