Hypoxia controls expression of kidney-pathogenic MUC1 variants.

The interplay between genetic and environmental factors influences the course of chronic kidney disease (CKD). In this context, genetic alterations in the kidney disease gene MUC1 (Mucin1) predispose to the development of CKD. These variations comprise the polymorphism rs4072037, which alters splicing of MUC1 mRNA, the length of a region with variable number of tandem repeats (VNTR), and rare autosomal-dominant inherited dominant-negative mutations in or 5' to the VNTR that causes autosomal dominant tubulointerstitial kidney disease (ADTKD-MUC1). As hypoxia plays a pivotal role in states of acute and chronic kidney injury, we explored the effects of hypoxia-inducible transcription factors (HIF) on the expression of MUC1 and its pathogenic variants in isolated primary human renal tubular cells. We defined a HIF-binding DNA regulatory element in the promoter-proximal region of MUC1 from which hypoxia or treatment with HIF stabilizers, which were recently approved for an anti-anemic therapy in CKD patients, increased levels of wild-type MUC1 and the disease-associated variants. Thus, application of these compounds might exert unfavorable effects in patients carrying MUC1 risk variants.

Estrogen-mediated downregulation of HIF-1α signaling in B lymphocytes influences postmenopausal bone loss.

In the bone marrow, B cells and bone-resorbing osteoclasts colocalize and form a specific microenvironment. How B cells functionally influence osteoclasts and bone architecture is poorly understood. Using genetically modified mice and high-throughput analyses, we demonstrate that prolonged HIF-1α signaling in B cells leads to enhanced RANKL production and osteoclast formation. In addition, deletion of HIF-1α in B cells prevents estrogen deficiency-induced bone loss in mice. Mechanistically, estrogen controls HIF-1α protein stabilization through HSP70-mediated degradation in bone marrow B cells. The stabilization of HIF-1α protein in HSP70-deficient bone marrow B cells promotes RANKL production and osteoclastogenesis. Induction of HSP70 expression by geranylgeranylacetone (GGA) administration alleviates ovariectomy-induced osteoporosis. Moreover, RANKL gene expression has a positive correlation with HIF1A expression in human B cells. In conclusion, HIF-1α signaling in B cells is crucial for the control of osteoclastogenesis, and the HSP70/HIF-1α axis may serve as a new therapeutic target for osteoporosis.

Hypoxia-inducible factor-1α is a critical transcription factor for IL-10-producing B cells in autoimmune disease.

Hypoxia-inducible factors (HIFs) are key elements for controlling immune cell metabolism and functions. While HIFs are known to be involved in T cells and macrophages activation, their functions in B lymphocytes are poorly defined. Here, we show that hypoxia-inducible factor-1α (HIF-1α) contributes to IL-10 production by B cells. HIF-1α regulates IL-10 expression, and HIF-1α-dependent glycolysis facilitates CD1dhiCD5+ B cells expansion. Mice with B cell-specific deletion of Hif1a have reduced number of IL-10-producing B cells, which result in exacerbated collagen-induced arthritis and experimental autoimmune encephalomyelitis. Wild-type CD1dhiCD5+ B cells, but not Hif1a-deficient CD1dhiCD5+ B cells, protect recipient mice from autoimmune disease, while the protective function of Hif1a-deficient CD1dhiCD5+ B cells is restored when their defective IL-10 expression is genetically corrected. Taken together, this study demonstrates the key function of the hypoxia-associated transcription factor HIF-1α in driving IL-10 expression in CD1dhiCD5+ B cells, and in controlling their protective activity in autoimmune disease.

Hypoxia regulates the sperm associated antigen 4 (SPAG4) via HIF, which is expressed in renal clear cell carcinoma and promotes migration and invasion in vitro.

Hypoxia leads to the upregulation of a variety of genes mediated largely via the hypoxia inducible transcription factor (HIF). Prominent HIF-regulated target genes such as the vascular endothelial growth factor (VEGF), the glucose transporter 1 (Glut-1), or erythropoietin (EPO) help to assure survival of cells and organisms in a low oxygenated environment. Here, we are the first to report the hypoxic regulation of the sperm associated antigen 4 (SPAG4). SPAG4 is a member of the cancer testis (CT) gene family and to date little is known about its physiological function or its involvement in tumor biology. A number of CT family candidate genes are therefore currently being investigated as potential cancer markers, due to their predominant testicular expression pattern. We analyzed RNA and protein expression by RNAse protection assay, immunofluorescent as well as immunohistological stainings. To evaluate the influence of SPAG4 on migration and invasion capabilities, siRNA knockdown as well as transient overexpression was performed prior to scratch or invasion assay analysis. The hypoxic regulation of SPAG4 is clearly mediated in a HIF-1 and VHL dependent manner. We furthermore show upregulation of SPAG4 expression in human renal clear cell carcinoma (RCC) and co-localization within the nucleolus in physiological human testis tissue. SPAG4 knockdown reduces the invasion capability of RCC cells in vitro and overexpression leads to enhancement of tumor cell migration. Together, SPAG4 could possibly play a role in the invasion capability and growth of renal tumors and could represent an interesting target for clinical intervention.

HIF-1 or HIF-2 induction is sufficient to achieve cell cycle arrest in NIH3T3 mouse fibroblasts independent from hypoxia.

Hypoxia is a severe stress which induces physiological and molecular adaptations, where the latter is dominated by the Hypoxia-inducible transcription Factor (HIF). A well described response on cellular level upon exposure to hypoxia is a reversible cell cycle arrest, which probably renders the cells more resistant to the difficult environment. The individual roles of hypoxia itself and of the isoforms HIF-1alpha and HIF-2alpha in cell cycle regulation are poorly understood and discussed controversially. In order to characterize the isolated effect of both HIFalpha isoforms on the cell cycle we generated tetracycline inducible, HIF-1alpha and -2alpha expressing NIH3T3 cells. The cDNAs for HIFalpha were mutated to generate stable and active HIF under normoxia. Upon activation of both HIFalpha subunits, the total number of living cells was reduced and long-term stimulation of HIF led to complete loss of transgene expression, implicating a strong negative selection pressure. Equally, colony forming activity was reduced by activation of both HIFalpha subunits. Cell cycle analyses showed that HIF activation resulted in a prominent cell cycle arrest in G(1)-phase, similarly to the hypoxic effect. Both, HIF-1alpha and HIF-2alpha were able to induce the expression of the cyclin-dependent kinase inhibitor p27 on reporter gene and protein level. Our study shows that HIF-1 and HIF-2 can individually arrest the cell cycle independent from hypoxia. These findings have implications for the resistance of tumor cells to the environment and treatment, but also for physiological cells. Importantly, recent approaches to stabilize HIFalpha in normoxia could have deleterious effects on proliferating tissues.