Evidence for transcriptional regulation of the glucose-6-phosphate transporter by HIF-1alpha: Targeting G6PT with mumbaistatin analogs in hypoxic mesenchymal stromal cells

Abstract

Mesenchymal stromal cell (MSC) markers are expressed on brain tumor-initiating cells involved in the development of hypoxic glioblastoma. Given that MSCs can survive hypoxia and that the glucose-6-phosphate transporter (G6PT) provides metabolic control that contributes to MSC mobilization and survival, we investigated the effects of low oxygen (1.2% O(2)) exposure on G6PT gene expression. We found that MSCs significantly expressed G6PT and the glucose-6-phosphatase catalytic subunit beta, whereas expression of the glucose-6-phosphatase catalytic subunit alpha and the islet-specific glucose-6-phosphatase catalytic subunit-related protein was low to undetectable. Analysis of the G6PT promoter sequence revealed potential binding sites for hypoxia inducible factor (HIF)-1alpha and for the aryl hydrocarbon receptor (AhR) and its dimerization partner, the AhR nuclear translocator (ARNT), AhR:ARNT. In agreement with this, hypoxia and the hypoxia mimetic cobalt chloride induced the expression of G6PT, vascular endothelial growth factor (VEGF), and HIF-1alpha. Gene silencing of HIF-1alpha prevented G6PT and VEGF induction in hypoxic MSCs whereas generation of cells stably expressing HIF-1alpha resulted in increased endogenous G6PT gene expression. A semisynthetic analog of the polyketide mumbaistatin, a potent G6PT inhibitor, specifically reduced MSC-HIF-1alpha cell survival. Collectively, our data suggest that G6PT may account for the metabolic flexibility that enables MSCs to survive under conditions characterized by hypoxia and could be specifically targeted within developing tumors.

Fig.1. Murine MSC strongly express the G6PT component of the glucose-6-phosphatase system

Total RNA was extracted from mouse MSC, mouse liver, and from mouse pancreas as described in the Methods section. cDNA synthesis and semi-quantitative RT-PCR were performed to assess gene expression of the glucose-6-phosphate transporter (G6PT), the glucose-6-phosphatase catalytic subunit α (G6PC), the islet-specific glucose-6-phosphatase catalytic subunit-related protein (G6PC-2), and the glucose-6-phosphatase catalytic subunit β (G6PC-3).

Fig.2. Cobalt chloride-induced chemical hypoxia and hypoxic culture conditions regulate G6PT gene expression

(A) Sub-confluent MSC were serum-starved and cultured under normoxic (5% CO₂ and 95% air; white box), or hypoxic (1% O₂, 5% CO₂, and 94% N₂; black box) conditions, or treated with 100 µM CoCl₂ (grey box) for 18 hours. Total RNA was extracted and qRT-PCR performed in order to assess the G6PT, G6PC-3 HIF-1α, VEGF, and MT1-MMP gene expression levels. (B) A 1133 bp sequence upstream of the ATG coding sequence of the murine G6PT gene promoter sequence was analyzed (NCBI source NM_008063.2), and located on mouse chromosome 9 at location 44,205,182-44,211,045. The core consensus sequence of the hypoxia responsive elements (A_G)CGT(G_C)C is denoted by asterisks (*, 50, 51). Sequences of a TATA box (−152/−147) and potential binding sites for HIF-1 (−1122/−1114), AhR:Arnt (−1059/−1051 and −1045/−1035), HNF1 (−177/−164), HNF3 (−71/−65), and C/EBP (−48/−40) are boxed.

Fig.3. Gene silencing of HIF-1α antagonizes the effects of hypoxia on G6PT gene expression

(A) MSC were transiently transfected with scrambled sequences (Mock, white bars) or HIF-1α siRNA (black bars) as described in the Methods section. Cells were then cultured under normal or hypoxic culture conditions, total RNA was extracted and qRT-PCR was used to assess HIF-1α, VEGF, and MT1-MMP gene expression as described in Fig.2. (B) G6PT gene expression was assessed by qRT-PCR in Mock-transfected (open circles) and in siHIF-1α-(closed circles) transfected cells that were subsequently cultured under hypoxic conditions.

Fig.4. Constitutive expression of an oxygen-dependent degradation domain HIF-1α mutant triggers G6PT gene expression

(A) Basal migration of MSC and MSC stably expressing a deletion mutant of HIF-1α (HIF-1α ΔODD, MSC-HIF) was performed as described in the Methods section. (B) Total RNA was extracted from MSC (white bars) and MSC-HIF (black bars), and qRT-PCR performed to assess the gene expression levels of G6PT, G6PC-3, VEGF, HIF-1α, and MT1-MMP. (C) Nuclear extracts were islolated from MSC and MSC-HIF and Westernblotting performed to detect nuclear HIF-1α or nuclear poly-(ADP-ribose) polymerase (PARP) expression. (D) Electrophoretic mobility assays were performed as described in the Methods section using nuclear extracts (Nuc. Extr.) isolated from MSC-HIF. NSC, cold unrelated non-specific competitor; SC, cold HIF-1α specific competitor; Ab, HIF-1α (1 µg) blocking antibody.

Fig.5. The mumbaistatin analog and potent G6PT inhibitor AD4-015 specifically triggers cell death in MSC constitutively expressing HIF-1α ΔODD

(A) Chemical structures of chlorogenic acid (CHL) and mumbaistatin analog (AD4-015). (B) MSC or MSC-HIF were cultured under normoxic conditions then treated for 18 hrs with 25 µM AD4-015 (or respective DMSO vehicle), or with 100 µM CHL (or respective EtOH vehicle). Hoechst (apoptosis) and propidium iodide (necrosis/late apoptosis) cell labelling was then performed and visualised using fluorescence microscopy. (C) Quantification was performed by visual counting. The mean of 4 fields from 3 independent experiments is shown. Probability values of less than 0.05 were considered significant, and an asterisk (*) identifies such significance. (D) MSC and MSC-HIF were treated with AD4-015 as in (B) and lysates isolated. SDS-PAGE followed by immunodetection of phospho-Erk, phospho-Akt, or GAPDH was performed as described in the Methods section.