Crosstalk between Microbiota-Derived Short-Chain Fatty Acids and Intestinal Epithelial HIF Augments Tissue Barrier Function.

New Anemia Therapies: Translating Novel Strategies From Bench to Bedside

STRUCTURAL MODIFICATIONS OF BUTYRATE REVEAL TEMPLATES FOR INTESTINAL EPITHELIAL HIF STABILIZERS

The interactions between the enteric microbiota and distal gut play important roles in regulating human health. Short chain fatty acids (i.e. butyrate) are produced by anaerobic microbes and are a major energy source for the colonic epithelium. Decreased concentrations of butyrate is strongly associated with colonic disease. Interestingly, the low oxygen conditions that enable butyrate production also place unusual metabolic demands on the colonic mucosa. However, the colonic epithelium is uniquely adapted to this austere oxygen environment through the stabilization of hypoxia‐inducible factor (HIF) and expression of HIF target genes. Because anaerobes are the major producers of butyrate, we hypothesized that microbe‐derived butyrate regulates host HIF. Studies with human intestinal epithelial cells (IECs) revealed that butyrate promotes mitochondrial‐dependent oxygen consumption and stabilizes HIF1α. Furthermore, butyrate activated HIF target genes in IECs and antibiotics decreased butyrate and HIF expression in the mouse colon. Germ free mice also showed decreased HIF1α expression in the colonic epithelium. Finally, we found that butyrate rescues physiologic hypoxia and protects the intestinal barrier via HIF. This indicates that butyrate participates in a diverse set of functions in the gut, including regulation of barrier function, and could be a potential therapeutic for mucosal protection.

Targeting the Hypoxia-Adenosine Link for Controlling Excessive Inflammation.

The long-term clinical experiences with recombinant human erythropoietin (rHuEPO) and its analog derivatives have clearly proven that correction of anemia with erythropoiesis stimulating agent (ESA) not only reduces blood transfusion and improves patients’ QOL but has multiple benefits for the concurrent complications of CKD such as Cardio-Renal–Anemia (CRA) syndrome and/or malnutrition-inflammation-atherosclerosis (MIA) syndrome.Unlike ESA, the newly available agent, hypoxia-inducible factor (HIF) stabilizer, stimulates endogenous erythropoietin (EPO) by mimicking hypoxia with HIF prolyl hydroxylase domain enzyme (HIF-PHD) inhibition. The phase 2 and 3 clinical studies have shown that HIF stabilizers are as efficacious as ESA in ameliorating renal anemia. Whether the same clinical benefits on CRA and MIA syndrome hold true in patients given HIF stabilizers is a matter for future debate. Given that HIF stabilizers act on the multiple target genes, the use of this novel agent may lead to unwanted adverse events.Launching HIF stabilizers into the treatment of renal anemia provokes a concern about how this alternative treatment will be taken up in the daily clinical practice. However, guideline-oriented strategies on how to use HIF stabilizer is not available at this limited point due to scant clinical information. Nevertheless, this opinion-based review provides a future insight into the management of renal anemia with HIF stabilizer by reference to the past experiences with ESA. HIF stabilizers can preferably be indicated for CRA syndrome at pre-dialysis stage, ESA resistant anemia at advanced CKD stage, and perhaps for dysregulated iron metabolism akin to MIA syndrome in patients on dialysis.

Although the function of hypoxia-inducible factor 1 (HIF1) in many kinds of solid tumor has been revealed, the significance of HIF1 in osteosarcoma is still controversial and not well understood. Immunohistochemistry was used to detect HIF1 expression. The correlation between HIF1 and clinicopathology factors was analyzed by use of chi-squared tests. The prognostic value of HIF1 was evaluated by univariate and multivariate analysis. Moreover, the function of HIF1 in osteosarcoma cells was further investigated in in-vitro experiments by regulating HIF1 and vascular endothelial growth factor-A (VEGF-A) expression. Expression of HIF1 was high for 56.82 % of the samples in our investigation. HIF1 expression was significantly associated with positive metastasis (P = 0.037). By use of the Kaplan-Meier method, high expression of HIF1 was proved to be related to poorer overall survival (P = 0.007). By use of a Cox-regression model, HIF1 was identified as an independent prognostic biomarker (P = 0.019). We also proved that HIF1 can promote osteosarcoma invasion in hypoxia by inducing VEGF-A expression. HIF1 was identified as an independent prognostic biomarker in osteosarcoma. It can promote osteosarcoma cell invasion by inducing VEGF-A expression, indicating that HIF1 is a potential drug target in osteosarcoma.

Microbiota-derived short-chain fatty acids, including butyrate (BA), have multiple beneficial health effects. In the colon, BA concentrations range from 10 to 20 mM and up to 95% is utilized as energy by the mucosa. BA plays a key role in epithelial-barrier regulation and anti-inflammation, and regulates cell growth and differentiation, at least in part, due to its direct influence on stabilization of the transcription factor hypoxia-inducible factor (HIF). It remains unclear whether BA is the optimal metabolite for such a response. In this study, we explored metabolite mimicry as an attractive strategy for the biological response to HIF. We discovered that 4-mercapto butyrate (MBA) stabilizes HIF more potently and has a longer biological half-life than BA in intestinal epithelial cells (IECs). We validated the MBA-mediated HIF transcriptional activity through the induction of classic HIF gene targets in IECs and enhanced epithelial barrier formation in vitro. In-vivo studies with MBA revealed systemic HIF stabilization in mice, which was more potent than its parent BA metabolite. Mechanistically, we found that MBA enhances oxygen consumption and that the sulfhydryl group is essential for HIF stabilization, but exclusively as a four-carbon SCFA. These findings reveal a combined biochemical mechanism for HIF stabilization and provide a foundation for the discovery of potent metabolite-like scaffolds.

Short chain fatty acids and specifically butyrate (BA) have been well documented to show multiple beneficial influences on health. Typically, the concentration of colonic butyrate can range from 10‐20 mM and 95‐99% is metabolized into energy by the mucosa. BA plays a key role in epithelial barrier regulation, ameliorates inflammation and regulates cell growth and differentiation. There are multiple mechanisms on how it plays such an important role in gut health, many related to its regulatory capacity for gene expression. Our laboratory recently reported a direct influence of BA on the stabilization of the transcription factor hypoxia‐inducible factor (HIF). BA acts directly and non‐competitively inhibits HIF prolyl hydroxylase 2 (PHD2), responsible for the rapid degradation of HIF in normoxia (NX). However, the efficacy of BA on HIF stabilization is limited due to its rapid metabolism as fuel. Based on these observations, we hypothesize that analogues structurally related to butyrate stabilize HIF with a longer biological half‐life. A small library of BA derivatives was screened in search of a non‐endogenous analogue with higher potency and/or half‐life for HIF stabilization. In vitro screenings at a physiologically‐relevant concentration (5 mM) were performed in intestinal epithelial cell lines T84 and CaCo2 in NX and HIF‐1a ELISA was used to determine protein abundance. Several hits were observed, with 4‐mercapto‐butyrate (SHBA) being the most promising candidate. The best BA analogues were validated through the induction of classic HIF gene targets, including BNIP3 and CaIX by q‐PCR. SHBA exhibited higher induction of these targets compared to BA. Dose response and time‐course studies were performed to compare SHBA and BA. Notably, SHBA exhibits a much longer half‐life as observed in the stabilization of HIF and induction of gene targets at least up to 72 h compared to BA (24 h). In vivo studies in C57BL/6 mice explored the induction of classic HIF targets and specifically erythropoietin (EPO) in circulation following administration of BA and SHBA at similar doses. These studies revealed that induction of EPO with a single dose of SHBA exceeded that of native BA. Thus, a non‐endogenous BA derivative (SHBA) stabilizes HIF at a higher potency and longer half‐life than BA and will be useful as a template for the development of a HIF stabilizing agent.

The lack of oxygen at high altitudes presents constraints for mammals. One of the mechanisms involved in the response to hypoxia is the stabilization of HIF‐1 (Hypoxia Inducible Factor 1) which contributes to ventilatory and metabolic responses, reduces mitochondrial respiration and stimulates anaerobic glycolysis. Upon acute hypoxia exposure, the expression of HIF‐1 reaches peak levels after a few hours of hypoxic exposure. In rats, however, the expression of HIF‐1 in hypoxia appears to be limited, as are the ventilatory and metabolic responses in comparison to other species better adapted to altitude. The purpose of this study is to better understand the interactions between hypoxia and HIF‐1 and to determine if there are other mechanisms possibly related to hypoxic responses. To this end, we measured ventilation and metabolic rate under resting conditions (whole body plethysmography) and mitochondrial respiration of the cerebral cortex and liver (oxygraphy) in adult male SD rats exposed in normoxia (21% O2 ‐ 6h), in hypoxia (10% O2 ‐ 6h), in normoxia after injection of a HIF‐1 stabilizer (deferoxamine; 100mg/kg), and in hypoxia after injection of a HIF‐1 inhibitor (2‐methoxyestradiol; 5mg/kg). Hypoxia reduces metabolism, increases minute ventilation and respiratory rate by 171 and 59% respectively and increases cytochrome c oxidase (COX) activity by an average of 72% in the cortex. Stabilization of HIF‐1 in normoxia doesn't result in any of these responses. However, inhibition of HIF‐1 in hypoxia decreases ventilatory responses by 30% for minute ventilation and 23% for respiratory rate and completely blocks the increase in COX activity. HIF‐1 would therefore be totally responsible for the IV complex responses observed in hypoxia, but would only be partially involved in the ventilatory response. There thus appears to be an interaction between HIF‐1 and other factors induced in hypoxia to establish ventilatory responses.

Microbiota-derived short chain fatty acids, particularly butyrate (BA), show multiple beneficial influences on health. In the colon, BA ranges from 10-20 mM and up to 99% is utilized as a metabolic fuel by the mucosa. BA plays a key role in epithelial barrier regulation, reduces inflammation, and regulates cell growth and differentiation. There are multiple mechanisms by which BA contributes to gut health, many due to its regulatory capacity for gene expression. Our group reported (PMID: 34190032) a direct influence of BA on the stabilization of the transcription factor hypoxia-inducible factor (HIF). It is known that HIF stabilization is essential for appropriate mucosal barrier regulation and the coordination of regenerative capacity in the intestine. However, BA is constantly metabolized limiting its HIF stabilization effect. This observation led to the design and investigation of BA-mimicking compounds that stabilize HIF, but may not be involved in metabolism. We hypothesize that structural modifications of BA yield analogues that stabilize HIF with better efficacy and a longer biological half-life. A library of BA derivatives was screened in search of a non-endogenous analogue with higher potency and/or longer half-life for HIF stabilization. In vitro screenings at physiologically-relevant concentrations (5 mM) were performed using intestinal epithelial cell lines (T84 and CaCO2) in normoxia to determine HIF-1a protein abundance. Various analogues stabilized HIF, with 4-mercapto-butyrate (MBA) being the most promising candidate. The best analogues were validated through the induction of classic HIF gene targets, including BNIP3 and CAIX by q-PCR. MBA exhibited higher induction of these targets compared to BA. Time-course studies revealed that MBA exhibits significantly longer half-life as observed in the stabilization of HIF and induction of gene targets for up to 72 h compared to BA (24 h). Loss of gene induction was observed in MBA treated cells expressing lentiviral shRNA against HIF1b (HIF1b KD), supporting a HIF-dependent transcription. Furthermore, it’s been widely reported that BA enhances epithelial barrier, primarily through HIF coordinated barrier protection. Among several other BA derivatives, MBA enhanced and prolonged epithelial barrier function in intestinal epithelial cells. In vivo studies in C57BL/6 mice explored the induction of HIF targets in tissue and specifically erythropoietin (EPO) in circulation following administration of BA and MBA at similar doses. These studies revealed that induction of EPO with a single dose of MBA exceeded that of native BA. In colonic and kidney tissue, GLUT1 and BNIP3 were moderately induced in vivo. Thus, a non-endogenous BA derivative (MBA) stabilizes HIF at higher potency and longer half-life than native BA and will be used as a template for the development of HIF stabilizing agents.

The transcription factor hypoxia inducible factor-1 (HIF-1) is a master regulator of oxygen homeostasis in cells. HIF-1 consists of a hypoxia-dependent alpha (α) subunit and an oxygen-independent beta (β) subunit. HIF-1 activates transcription of over 100 genes involved in a wide range of cellular and molecular responses to ischemia and hypoxia including angiogenesis, glucose transport, glycolysis, antiapopotic and proapoptotic pathways, and cellular proliferation. In most tissues, HIF-1α is ubiquitinated and rapidly degraded under normoxic conditions, but stabilized from degradation and activated by hypoxia. Previously we have demonstrated that HIF-1 is abundant in Leydig cells and that, unlike HIF-1 in other tissues, testicular HIF-1 exists in a non-ubiquitinated form that is constitutively expressed in both the normoxic and hypoxic rat testis. We hypothesize that HIF-1 plays key roles in protecting Leydig cells from apoptosis and that HIF-1 is important for regulating oxygen microenvironments in the testis. Studies in our laboratory have focused on the role of HIF-1 in the normoxic testis and in conditions of testis ischemia such as testicular torsion. Prolyl hydroxylases 1 and 2 (PHD1 and PHD2) and Factor Inhibiting HIF (FIH1) are oxygen-dependent enzymes that hydroxylate the HIF-1α subunit under normoxic conditions. Hydroxylated HIF-1α binds to the von Hippel-Lindau (pVHL) tumor suppressor protein, a ubiquitin ligase that targets HIF-1α for ubiquitination and subsequent degradation by the proteasome. The purpose of this study was to examine expression of PHDs, FIH1, and pVHL in the rat testis as an initial step towards understanding the regulatory mechanisms responsible for constitutive expression of testicular HIF-1. Experimental unilateral testicular ischemia (I) was created by 720° of testicular torsion for 1h followed by 4h or 24h of reperfusion (I/R). Cytoplasmic and nuclear proteins were isolated from sham-operated and ischemic testes and steady-state levels of PHD1, PHD2, FIH-1, and pVHL proteins detected by immunoblot analysis. PHD1 and 2 were constitutively expressed in normoxic and ischemic testes (P<0.05), and PHD2 was the predominant isoform detected. FIH1 and pVHL were also abundant in the testis and the levels of these proteins showed no significant changes following I or I/R (P<0.05). In conclusion, constitutive expression of HIF-1 and HIF-1 regulatory proteins in the normoxic and ischemic testis suggest that stabilization and activation of HIF-1 in the testis may occur through pathways that are fundamentally different from HIF-1 activation mechanisms in other tissues. Further studies on the functions of testicular HIF-1 and HIF-1 regulatory pathways will advance an understanding of the role of Leydig cells in oxygen homeostasis of the testis and cellular and molecular responses to testis ischemia and hypoxia. Supported by NIH grant R15HD046451 to M.A.P.

The gastrointestinal tract is home to trillions of microorganisms that interact with their host in profound ways, including regulation of immune, endocrine, and neurological functions. One mechanism by which these microbes interact with their eukaryotic host is through the generation of short-chain fatty acids (SCFAs), which are metabolized by the intestinal epithelium creating a state of "physiologic hypoxia". This hypoxia, in turn, results in stabilization and activation of hypoxia-inducible factor (HIF), a transcription factor family shown to support gut barrier function and homeostasis, in the intestinal epithelium. The association between HIF and intestinal homeostasis has been long understood, as both genetic and pharmacologic potentiation of the HIF signaling pathway has been shown to promote barrier function both in vitro and in vivo . Although it has been previously established that pathogenic bacteria regulate HIF stabilization and activity in the intestinal epithelium independent of SCFA metabolism, it is not clear whether this property extends to noninfectious and/or commensal bacterial species. Here, we demonstrate that nonpathogenic, commensal strains of Escherichia coli stabilize HIF in intestinal epithelial cells in vitro . Further, we show that HIF is transcriptionally active in these cells and drives a "pro-barrier" transcriptional program. This property was found to be dependent on bacterial aerobic respiration, as genetic elimination of E. coli aerobic respiration abolished HIF stabilization and the subsequent transcriptional phenotype. Finally, we observed induction of tissue hypoxia in vivo using antibiotic-treated mice colonized with wild-type, but not respiration-deficient, E. coli. These findings demonstrate a novel ability for probiotic E. coli to regulate intestinal homeostasis through activation of HIF and suggest that this mechanism might be leveraged in as a novel therapeutic to combat intestinal inflammation, such as that observed during inflammatory bowel disease (IBD).

Hypoxia inducible factor stabilization improves defective ischemia-induced angiogenesis in a rodent model of chronic kidney disease

Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric basic helix-loop-helix transcription factor composed of HIF-1 alpha and HIF-1 beta/aryl hydrocarbon nuclear translocator subunits. HIF-1 expression is induced by hypoxia, growth factors, and activation of oncogenes. In response to hypoxia, HIF-1 activates the expression of many genes including vascular endothelial growth factor (VEGF) and erythropoietin. HIF-1 and VEGF play an important role in angiogenesis and tumor progression. Vanadate is widely used in industry, and is a potent inducer of tumors in humans and animals. In this study, we demonstrate that vanadate induces HIF-1 activity through the expression of HIF-1alpha but not HIF-1 beta subunit, and increases VEGF expression in DU145 human prostate carcinoma cells. We also studied the signaling pathway involved in vanadate-induced HIF-1 alpha and VEGF expression and found that phosphatidylinositol 3-kinase/Akt signaling was required for HIF-1 and VEGF expression induced by vanadate, whereas mitogen-activated protein kinase pathway was not required. We also found that reactive oxygen species (ROS) were involved in vanadate-induced expression of HIF-1 and VEGF in DU145 cells. The major species of ROS responsible for the induction of HIF-1 and VEGF expression was H(2)O(2). These results suggest that the expression of HIF-1 and VEGF induced by vanadate through PI3K/Akt may be an important signaling pathway in the vanadate-induced carcinogenesis, and ROS may play an important role.

Breast cancer is one of the most common cancers among women. Many factors are associated with this disease including overexpression/amplification of the tyrosine kinase HER2/ErbB2. ErbB2 belongs to the EGFR-family of receptor tyrosine kinases and its overexpression can activate multiple signaling pathways that can promote tumor progression via regulation of cell growth, proliferation, metabolism, and survival. Breast cancer patients with ErbB2 amplifications are currently treated with lapatinib (Tykerb), a small-molecule kinase inhibitor that specifically blocks the active site of ErbB2 to inhibit EGFR/ErbB2 downstream signaling. While lapatinib has displayed clinical efficacy in breast cancer patients, acquired resistance has been reported and the exact mechanism of this resistance is poorly understood. Hypoxia or low oxygen tension occurs in many breast cancers and is associated with poor patient survival. One of the major players in the cellular response to hypoxia is stabilization of the transcription factor hypoxia inducible factor 1 (HIF-1). HIF-1 is a transcription factor that regulates cell growth, proliferation and survival. As a result, hypoxic tumors often exhibit weak response to different chemotherapeutic agents and poor prognosis. Here, we show that hypoxia, via HIF-1α, blocks lapatinib-mediated effects on ErbB2-expressing mammary epithelial and ErbB2-positive breast cancer cells. Lapatinib-mediated growth inhibition is reduced under hypoxic conditions (1%O2). Consistent with these results, we find that hypoxia can maintain activation of signaling pathways downstream from ErbB2 including AKT and ERK in the presence of lapatinib. This protective effect depends on HIF-1 expression as cells expressing HIF-1 RNAi no longer are resistant to lapatinib treatment. Moreover, overexpression of stable HIF-1 in ErbB2-expressing cells under normoxic conditions is sufficient to inhibit lapatinib-mediated effects on proliferation and survival and maintain ERK and AKT signaling. One possible mechanism of how HIF-1 may bypass lapatinib-treated inhibition of the ERK pathway is via regulation of dual-specificity phosphatase 2 (DUSP2). DUSP2 is a negative regulator of the ERK pathway and we show that cells exposed to hypoxia contain decreased DUSP2 levels and correlate with increased Erk activation. Indeed, reducing DUSP2 expression alone in ErbB2-positive breast cancer cells is sufficient to reduce lapatinib-mediated effects. Thus, hypoxia, via HIF-1, causes lapatinib resistance in ErbB2-expressing breast cancer cells and suggests that targeting ERK/MAPK pathway in hypoxic breast cancer may increase sensitivity to ErbB2-targeted therapy. This abstract is also presented as poster C63. Citation Format: Sergey V. Karakashev, Reginato J. Mauricio. Hypoxia induces lapatinib resistance in ErbB2-positive breast cancer cells via regulation of DUSP2. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr PR15.