Ectopic MYBL2-Mediated Regulation of Androglobin Gene Expression.

Androglobin (ADGB) is a highly conserved and recently identified member of the globin superfamily. Although previous studies revealed a link to ciliogenesis and an involvement in murine spermatogenesis, its physiological function remains mostly unknown. Apart from FOXJ1-dependent regulation, the transcriptional landscape of the ADGB gene remains unexplored. We, therefore, aimed to obtain further insights into regulatory mechanisms governing ADGB expression. To this end, changes in ADGB promoter activity were examined using luciferase reporter gene assays in the presence of a set of more than 475 different exogenous transcription factors. MYBL2 and PITX2 resulted in the most pronounced increase in ADGB promoter-dependent luciferase activity. Subsequent truncation strategies of the ADGB promoter fragment narrowed down the potential MYBL2 and PITX2 binding sites within the proximal ADGB promoter. Furthermore, MYBL2 binding sites on the ADGB promoter were further validated via a guide RNA-mediated interference strategy using reporter assays. Chromatin immunoprecipitation (ChIP)-qPCR experiments illustrated enrichment of the endogenous ADGB promoter region upon MYBL2 and PITX2 overexpression. Consistently, ectopic MYBL2 expression induced endogenous ADGB mRNA levels. Collectively, our data indicate that ADGB is strongly regulated at the transcriptional level and might have functions beyond ciliogenesis.

Analysis of the Hypoxic Response in a Mouse Cortical Collecting Duct-Derived Cell Line Suggests That Esrra Is Partially Involved in Hif1α-Mediated Hypoxia-Inducible Gene Expression in mCCDcl1 Cells.

The kidney is strongly dependent on a continuous oxygen supply, and is conversely highly sensitive to hypoxia. Controlled oxygen gradients are essential for renal control of solutes and urine-concentrating mechanisms, which also depend on various hormones including aldosterone. The cortical collecting duct (CCD) is part of the aldosterone-sensitive distal nephron and possesses a key function in fine-tuned distal salt handling. It is well known that aldosterone is consistently decreased upon hypoxia. Furthermore, a recent study reported a hypoxia-dependent down-regulation of sodium currents within CCD cells. We thus investigated the possibility that cells from the cortical collecting duct are responsive to hypoxia, using the mouse cortical collecting duct cell line mCCDcl1 as a model. By analyzing the hypoxia-dependent transcriptome of mCCDcl1 cells, we found a large number of differentially-expressed genes (3086 in total logFC< −1 or >1) following 24 h of hypoxic conditions (0.2% O2). A gene ontology analysis of the differentially-regulated pathways revealed a strong decrease in oxygen-linked processes such as ATP metabolic functions, oxidative phosphorylation, and cellular and aerobic respiration, while pathways associated with hypoxic responses were robustly increased. The most pronounced regulated genes were confirmed by RT-qPCR. The low expression levels of Epas1 under both normoxic and hypoxic conditions suggest that Hif-1α, rather than Hif-2α, mediates the hypoxic response in mCCDcl1 cells. Accordingly, we generated shRNA-mediated Hif-1α knockdown cells and found Hif-1α to be responsible for the hypoxic induction of established hypoxically-induced genes. Interestingly, we could show that following shRNA-mediated knockdown of Esrra, Hif-1α protein levels were unaffected, but the gene expression levels of Egln3 and Serpine1 were significantly reduced, indicating that Esrra might contribute to the hypoxia-mediated expression of these and possibly other genes. Collectively, mCCDcl1 cells display a broad response to hypoxia and represent an adequate cellular model to study additional factors regulating the response to hypoxia.

Androglobin, a chimeric mammalian globin, is required for male fertility.

Spermatogenesis is a highly specialized differentiation process driven by a dynamic gene expression program and ending with the production of mature spermatozoa. Whereas hundreds of genes are known to be essential for male germline proliferation and differentiation, the contribution of several genes remains uncharacterized. The predominant expression of the latest globin family member, androglobin (Adgb), in mammalian testis tissue prompted us to assess its physiological function in spermatogenesis. Adgb knockout mice display male infertility, reduced testis weight, impaired maturation of elongating spermatids, abnormal sperm shape, and ultrastructural defects in microtubule and mitochondrial organization. Epididymal sperm from Adgb knockout animals display multiple flagellar malformations including coiled, bifid or shortened flagella, and erratic acrosomal development. Following immunoprecipitation and mass spectrometry, we could identify septin 10 (Sept10) as interactor of Adgb. The Sept10-Adgb interaction was confirmed both in vivo using testis lysates and in vitro by reciprocal co-immunoprecipitation experiments. Furthermore, the absence of Adgb leads to mislocalization of Sept10 in sperm, indicating defective manchette and sperm annulus formation. Finally, in vitro data suggest that Adgb contributes to Sept10 proteolysis in a calmodulin-dependent manner. Collectively, our results provide evidence that Adgb is essential for murine spermatogenesis and further suggest that Adgb is required for sperm head shaping via the manchette and proper flagellum formation.

Androglobin gene expression patterns and FOXJ1-dependent regulation indicate its functional association with ciliogenesis.

Androglobin (ADGB) represents the latest addition to the globin superfamily in metazoans. The chimeric protein comprises a calpain domain and a unique circularly permutated globin domain. ADGB expression levels are most abundant in mammalian testis, but its cell-type-specific expression, regulation, and function have remained unexplored. Analyzing bulk and single-cell mRNA-Seq data from mammalian tissues, we found that-in addition to the testes-ADGB is prominently expressed in the female reproductive tract, lungs, and brain, specifically being associated with cell types forming motile cilia. Correlation analysis suggested coregulation of ADGB with FOXJ1, a crucial transcription factor of ciliogenesis. Investigating the transcriptional regulation of the ADGB gene, we characterized its promoter using epigenomic datasets, exogenous promoter-dependent luciferase assays, and CRISPR/dCas9-VPR-mediated activation approaches. Reporter gene assays revealed that FOXJ1 indeed substantially enhanced luciferase activity driven by the ADGB promoter. ChIP assays confirmed binding of FOXJ1 to the endogenous ADGB promoter region. We dissected the minimal sequence required for FOXJ1-dependent regulation and fine mapped the FOXJ1 binding site to two evolutionarily conserved regions within the ADGB promoter. FOXJ1 overexpression significantly increased endogenous ADGB mRNA levels in HEK293 and MCF-7 cells. Similar results were observed upon RFX2 overexpression, another key transcription factor in ciliogenesis. The complex transcriptional regulation of the ADGB locus was illustrated by identifying a distal enhancer, responsible for synergistic regulation by RFX2 and FOXJ1. Finally, cell culture studies indicated an ADGB-dependent increase in the number of ciliated cells upon overexpression of the full-length protein, confirming a ciliogenesis-associated role of ADGB in mammals.

Lessons from the post-genomic era: Globin diversity beyond oxygen binding and transport.

Vertebrate hemoglobin (Hb) and myoglobin (Mb) were among the first proteins whose structures and sequences were determined over 50 years ago. In the subsequent pregenomic period, numerous related proteins came to light in plants, invertebrates and bacteria, that shared the myoglobin fold, a signature sequence motif characteristic of a 3-on-3 α-helical sandwich. Concomitantly, eukaryote and bacterial globins with a truncated 2-on-2 α-helical fold were discovered. Genomic information over the last 20 years has dramatically expanded the list of known globins, demonstrating their existence in a limited number of archaeal genomes, a majority of bacterial genomes and an overwhelming majority of eukaryote genomes. In vertebrates, 6 additional globin types were identified, namely neuroglobin (Ngb), cytoglobin (Cygb), globin E (GbE), globin X (GbX), globin Y (GbY) and androglobin (Adgb). Furthermore, functions beyond the familiar oxygen transport and storage have been discovered within the vertebrate globin family, including NO metabolism, peroxidase activity, scavenging of free radicals, and signaling functions. The extension of the knowledge on globin functions suggests that the original roles of bacterial globins must have been enzymatic, involved in defense against NO toxicity, and perhaps also as sensors of O2, regulating taxis away or towards high O2 concentrations. In this review, we aimed to discuss the evolution and remarkable functional diversity of vertebrate globins with particular focus on the variety of non-canonical expression sites of mammalian globins and their according impressive variability of atypical functions.

Deletion of the serine protease CAP2/Tmprss4 leads to dysregulated renal water handling upon dietary potassium depletion.

The kidney needs to adapt daily to variable dietary K+ contents via various mechanisms including diuretic, acid-base and hormonal changes that are still not fully understood. In this study, we demonstrate that following a K+-deficient diet in wildtype mice, the serine protease CAP2/Tmprss4 is upregulated in connecting tubule and cortical collecting duct and also localizes to the medulla and transitional epithelium of the papilla and minor calyx. Male CAP2/Tmprss4 knockout mice display altered water handling and urine osmolality, enhanced vasopressin response leading to upregulated adenylate cyclase 6 expression and cAMP overproduction, and subsequently greater aquaporin 2 (AQP2) and Na+-K+-2Cl- cotransporter 2 (NKCC2) expression following K+-deficient diet. Urinary acidification coincides with significantly increased H+,K+-ATPase type 2 (HKA2) mRNA and protein expression, and decreased calcium and phosphate excretion. This is accompanied by increased glucocorticoid receptor (GR) protein levels and reduced 11ß-hydroxysteroid dehydrogenase 2 activity in knockout mice. Strikingly, genetic nephron-specific deletion of GR leads to the mirrored phenotype of CAP2/Tmprss4 knockouts, including increased water intake and urine output, urinary alkalinisation, downregulation of HKA2, AQP2 and NKCC2. Collectively, our data unveil a novel role of the serine protease CAP2/Tmprss4 and GR on renal water handling upon dietary K+ depletion.

Altered Prostasin (CAP1/Prss8) Expression Favors Inflammation and Tissue Remodeling in DSS-induced Colitis.

BACKGROUND: Inflammatory bowel diseases (IBD) including ulcerative colitis and Crohn's disease are diseases with impaired epithelial barrier function. We aimed to investigate whether mutated prostasin and thus, reduced colonic epithelial sodium channel activity predisposes to develop an experimentally dextran sodium sulfate (DSS)-induced colitis. METHODS: Wildtype, heterozygous (fr/+), and homozygous (fr/fr) prostasin-mutant rats were treated 7 days with DSS followed by 7 days of recovery and analyzed with respect to histology, clinicopathological parameters, inflammatory marker mRNA transcript expression, and sodium transporter protein expression. RESULTS: In this study, a more detailed analysis on rat fr/fr colons revealed reduced numbers of crypt and goblet cells, and local angiodysplasia, as compared with heterozygous (fr/+) and wildtype littermates. Following 2% DSS treatment for 7 days followed by 7 days recovery, fr/fr animals lost body weight, and reached maximal diarrhea score and highest disease activity after only 3 days, and strongly increased cytokine levels. The histology score significantly increased in all groups, but fr/fr colons further displayed pronounced histological alterations with near absence of goblet cells, rearrangement of the lamina propria, and presence of neutrophils, eosinophils, and macrophages. Additionally, fr/fr colons showed ulcerations and edemas that were absent in fr/+ and wildtype littermates. Following recovery, fr/fr rats reached, although significantly delayed, near-normal diarrhea score and disease activity, but exhibited severe architectural remodeling, despite unchanged sodium transporter protein expression. CONCLUSIONS: In summary, our results demonstrate a protective role of colonic prostasin expression against experimental colitis, and thus represent a susceptibility gene in the development of inflammatory bowel disease.

The Proteolytic Activation of (H3N2) Influenza A Virus Hemagglutinin Is Facilitated by Different Type II Transmembrane Serine Proteases.

UNLABELLED: Cleavage of influenza virus hemagglutinin (HA) by host cell proteases is necessary for viral activation and infectivity. In humans and mice, members of the type II transmembrane protease family (TTSP), e.g., TMPRSS2, TMPRSS4, and TMPRSS11d (HAT), have been shown to cleave influenza virus HA for viral activation and infectivity in vitro Recently, we reported that inactivation of a single HA-activating protease gene,Tmprss2, in knockout mice inhibits the spread of H1N1 influenza viruses. However, after infection of Tmprss2 knockout mice with an H3N2 influenza virus, only a slight increase in survival was observed, and mice still lost body weight. In this study, we investigated an additional trypsin-like protease, TMPRSS4. Both TMPRSS2 and TMPRSS4 are expressed in the same cell types of the mouse lung. Deletion of Tmprss4 alone in knockout mice does not protect them from body weight loss and death upon infection with H3N2 influenza virus. In contrast,Tmprss2(-/-)Tmprss4(-/-)double-knockout mice showed a remarkably reduced virus spread and lung pathology, in addition to reduced body weight loss and mortality. Thus, our results identified TMPRSS4 as a second host cell protease that, in addition to TMPRSS2, is able to activate the HA of H3N2 influenza virus in vivo IMPORTANCE: Influenza epidemics and recurring pandemics are responsible for significant global morbidity and mortality. Due to high variability of the virus genome, resistance to available antiviral drugs is frequently observed, and new targets for treatment of influenza are needed. Host cell factors essential for processing of the virus hemagglutinin represent very suitable drug targets because the virus is dependent on these host factors for replication. We reported previously that Tmprss2-deficient mice are protected against H1N1 virus infections, but only marginal protection against H3N2 virus infections was observed. Here we show that deletion of two host protease genes,Tmprss2 and Tmprss4, strongly reduced viral spread as well as lung pathology and resulted in increased survival after H3N2 virus infection. Thus, TMPRSS4 represents another host cell factor that is involved in cleavage activation of H3N2 influenza viruses in vivo.

Epithelial Sodium Channel-Mediated Sodium Transport Is Not Dependent on the Membrane-Bound Serine Protease CAP2/Tmprss4.

The membrane-bound serine protease CAP2/Tmprss4 has been previously identified in vitro as a positive regulator of the epithelial sodium channel (ENaC). To study its in vivo implication in ENaC-mediated sodium absorption, we generated a knockout mouse model for CAP2/Tmprss4. Mice deficient in CAP2/Tmprss4 were viable, fertile, and did not show any obvious histological abnormalities. Unexpectedly, when challenged with sodium-deficient diet, these mice did not develop any impairment in renal sodium handling as evidenced by normal plasma and urinary sodium and potassium electrolytes, as well as normal aldosterone levels. Despite minor alterations in ENaC mRNA expression, we found no evidence for altered proteolytic cleavage of ENaC subunits. In consequence, ENaC activity, as monitored by the amiloride-sensitive rectal potential difference (ΔPD), was not altered even under dietary sodium restriction. In summary, ENaC-mediated sodium balance is not affected by lack of CAP2/Tmprss4 expression and thus, does not seem to directly control ENaC expression and activity in vivo.

Mutations of the serine protease CAP1/Prss8 lead to reduced embryonic viability, skin defects, and decreased ENaC activity.

CAP1/Prss8 is a membrane-bound serine protease involved in the regulation of several different effectors, such as the epithelial sodium channel ENaC, the protease-activated receptor PAR2, the tight junction proteins, and the profilaggrin polypeptide. Recently, the V170D and the G54-P57 deletion mutations within the CAP1/Prss8 gene, identified in mouse frizzy (fr) and rat hairless (fr(CR)) animals, respectively, have been proposed to be responsible for their skin phenotypes. In the present study, we analyzed those mutations, revealing a change in the protein structure, a modification of the glycosylation state, and an overall reduction in the activation of ENaC of the two mutant proteins. In vivo analyses demonstrated that both fr and fr(CR) mutant animals present analogous reduction of embryonic viability, similar histologic aberrations at the level of the skin, and a significant decrease in the activity of ENaC in the distal colon compared with their control littermates. Hairless rats additionally had dehydration defects in skin and intestine and significant reduction in the body weight. In conclusion, we provided molecular and functional evidence that CAP1/Prss8 mutations are accountable for the defects in fr and fr(CR) animals, and we furthermore demonstrate a decreased function of the CAP1/Prss8 mutant proteins. Therefore, fr and fr(CR) animals are suitable models to investigate the consequences of CAP1/Prss8 action on its target proteins in the whole organism.