Analysis of Tumor-Associated AXIN1 Missense Mutations Identifies Variants That Activate ß-Catenin Signaling.

AXIN1 is a major component of the ß-catenin destruction complex and is frequently mutated in various cancer types, particularly liver cancers. Truncating AXIN1 mutations are recognized to encode a defective protein that leads to ß-catenin stabilization, but the functional consequences of missense mutations are not well characterized. Here, we first identified the GSK3ß, ß-catenin, and RGS/APC interaction domains of AXIN1 that are the most critical for proper ß-catenin regulation. Analysis of 80 tumor-associated variants in these domains identified 18 that significantly affected ß-catenin signaling. Coimmunoprecipitation experiments revealed that most of them lost binding to the binding partner corresponding to the mutated domain. A comprehensive protein structure analysis predicted the consequences of these mutations, which largely overlapped with the observed effects on ß-catenin signaling in functional experiments. The structure analysis also predicted that loss-of-function mutations within the RGS/APC interaction domain either directly affected the interface for APC binding or were located within the hydrophobic core and destabilized the entire structure. In addition, truncated AXIN1 length inversely correlated with the ß-catenin regulatory function, with longer proteins retaining more functionality. These analyses suggest that all AXIN1-truncating mutations at least partially affect ß-catenin regulation, whereas this is only the case for a subset of missense mutations. Consistently, most colorectal and liver cancers carrying missense variants acquire mutations in other ß-catenin regulatory genes such as APC and CTNNB1. These results will aid the functional annotation of AXIN1 mutations identified in large-scale sequencing efforts or in individual patients. SIGNIFICANCE: Characterization of 80 tumor-associated missense variants of AXIN1 reveals a subset of 18 mutations that disrupt its ß-catenin regulatory function, whereas the majority are passenger mutations.

Investigating theobromine as a potential anti-human coronaviral agent.

Coronaviruses (CoVs) have long been known to infect humans, mainly alpha-CoV and beta-CoV. The vaccines developed for SARS-CoV-2 are likely not effective against other coronavirus species, whereas the risk of the emergence of new strains that may cause the next epidemic/pandemic is high. The development of antiviral drugs that are effective across different CoVs represents a viable strategy for improving pandemic preparedness. In this study, we aim to identify pan-coronaviral agents by targeting the conserved main protease (Mpro). For drug screening, the catalytic dyad of four human CoVs (HCoVs: SARS-CoV-2, and seasonal CoV NL63, OC43, and 229E) was targeted by molecular docking. The identified leading candidate theobromine, a xanthine derivative, was further tested in cell culture models of coronavirus infection. Theobromine binds strongly with the catalytic dyad (His41 and Cys144/145) of SARS-CoV-2 and HCoV-NL63 Mpro, mildly with HCoV-OC43, but not with HCoV-229E. However, theobromine only shows dose-dependent inhibition in Calu3 cells inoculated with SARS-CoV-2, but not in cells inoculated with seasonal CoVs. Theobromine exerts antiviral activity against coronavirus infections potentially through targeting Mpro. However, the antiviral potency is distinct among different CoVs.

Immunosuppressants exert differential effects on pan-coronavirus infection and distinct combinatory antiviral activity with molnupiravir and nirmatrelvir.

BACKGROUND: Immunocompromised populations, such as organ transplant recipients and patients with inflammatory bowel disease (IBD) receiving immunosuppressive/immunomodulatory medications, may be more susceptible to coronavirus infections. However, little is known about how immunosuppressants affect coronavirus replication and their combinational effects with antiviral drugs. OBJECTIVE: This study aims to profile the effects of immunosuppressants and the combination of immunosuppressants with oral antiviral drugs molnupiravir and nirmatrelvir on pan-coronavirus infection in cell and human airway organoids (hAOs) culture models. METHODS: Different coronaviruses (including wild type, delta and omicron variants of SARS-CoV-2, and NL63, 229E and OC43 seasonal coronaviruses) were used in lung cell lines and hAOs models. The effects of immunosuppressants were tested. RESULTS: Dexamethasone and 5-aminosalicylic acid moderately stimulated the replication of different coronaviruses. Mycophenolic acid (MPA), 6-thioguanine (6-TG), tofacitinib and filgotinib treatment dose-dependently inhibited viral replication of all tested coronaviruses in both cell lines and hAOs. The half maximum effective concentration (EC50) of tofacitinib against SARS-CoV-2 was 0.62 µM and the half maximum cytotoxic concentration (CC50) was above 30 µM, which resulted in a selective index (SI) of about 50. The anti-coronavirus effect of the JAK inhibitors tofacitinib and filgotinib is dependent on the inhibition of STAT3 phosphorylation. Combinations of MPA, 6-TG, tofacitinib, and filgotinib with the oral antiviral drugs molnupiravir or nirmatrelvir exerted an additive or synergistic antiviral activity. CONCLUSIONS: Different immunosuppressants have distinct effects on coronavirus replication, with 6-TG, MPA, tofacitinib and filgotinib possessing pan-coronavirus antiviral activity. The combinations of MPA, 6-TG, tofacitinib and filgotinib with antiviral drugs exerted an additive or synergistic antiviral activity. Thus, these findings provide an important reference for optimal management of immunocompromised patients infected with coronaviruses.

Issues with RNF43 antibodies to reliably detect intracellular location.

RNF43 is an important negative regulator of ß-catenin signaling by removing Wnt-receptors from the membrane. It is often mutated in cancers, leading to aberrant Wnt-dependent nuclear translocation of ß-catenin. RNF43 has also been suggested to regulate ß-catenin signaling directly within the nucleus, among other proposed nuclear functions. Given the importance of RNF43 in regulating Wnt/ß-catenin signaling and its potential therapeutic relevance, a proper understanding of RNF43 biology is required. However, the presumed nuclear location is mainly based on available antibodies. These same antibodies have also been used extensively for immunoblotting or immunohistochemical purposes. However, a proper evaluation of their quality to reliably detect endogenous RNF43 has not been performed. Here, using genome editing we have generated a cell line that entirely misses RNF43 exons 8 and 9, encoding the epitopes of commonly used RNF43 antibodies. Using this clone in addition to various other cell line tools, we show that four RNF43 antibodies only yield non-specific signals when applied in immunoblotting, immunofluorescence and immunohistochemical experiments. In other words, they cannot reliably detect endogenous RNF43. Our results suggest that the nuclear staining patterns are an antibody artifact and that RNF43 is unlikely to localize within the nucleus. More generally, reports using RNF43 antibodies should be interpreted with caution, at least for the RNF43 protein aspects described in these papers.

Nirmatrelvir exerts distinct antiviral potency against different human coronaviruses.

Nirmatrelvir is the main component of Paxlovid, an oral antiviral drug approved for the treatment of COVID-19 caused by SARS-COV-2 infection. Nirmatrelvir targets the main protease (Mpro), which is substantially conserved among different coronaviruses. Here, our molecular docking analysis indicates comparable affinity of nirmatrelvir binding to the Mpro enzymes of SARS-CoV-2 and three seasonal coronaviruses (OC43, 229E and NL63). However, in cell culture models, we found that nirmatrelvir potently inhibited SARS-CoV-2, OC43 and 229E, but not NL63. The insensitivity of NL63 to nirmatrelvir treatment was demonstrated at both viral replication and infectious titer levels. The antiviral activity of nirmatrelvir against OC43 and 229E was further confirmed in human airway organoids. The combination of nirmatrelvir and molnupiravir exerted differential patterns of antiviral response against OC43 and 229E. These results revealed disparities in the ability of nirmatrelvir to inhibit different coronaviruses, and caution against repurposing of nirmatrelvir as a pan-coronavirus treatment.

Dissecting the multifaceted impact of statin use on fatty liver disease: a multidimensional study.

BACKGROUND: Statin use could benefit patients with non-alcoholic fatty liver disease (NAFLD), but the evidence is segmented and inconclusive. This multidimensional study comprehensively investigated the potential benefits and mechanism-of-action of statins in NAFLD. METHODS: A cross-sectional investigation was performed within the Rotterdam Study (general population; n = 4.576) and the PERSONS cohort (biopsy-proven NAFLD patients; n = 569). Exclusion criteria were secondary causes for steatosis and insufficient data on alcohol, dyslipidemia or statin use. Associations of statin use with NAFLD (among entire general population), fibrosis and NASH (among NAFLD individuals and patients) were quantified. These results were pooled with available literature in meta-analysis. Last, we assessed statins' anti-lipid and anti-inflammatory effects in 3D cultured human liver organoids and THP-1 macrophages, respectively. FINDINGS: Statin use was inversely associated with NAFLD in the Rotterdam study compared to participants with untreated dyslipidemia. In the PERSONS cohort, statin use was inversely associated with NASH, but not with fibrosis. The meta-analysis included 7 studies and indicated a not significant inverse association for statin use with NAFLD (pooled-Odds Ratio: 0.69, 95% Confidence Interval: 0.46-1.01) and significant inverse associations with NASH (pooled-OR: 0.59, 95% CI: 0.44-0.79) and fibrosis (pooled-OR: 0.48, 95% CI: 0.33-0.70). In vitro, statins significantly reduced lipid droplet accumulation in human liver organoids and downregulated expression of pro-inflammatory cytokines in macrophages. INTERPRETATION: Pooled results demonstrated that statin use was associated with a lower prevalence of NASH and fibrosis and might prevent NAFLD. This may be partially attributed to the anti-lipid and anti-inflammatory characteristics of statins. Given their under-prescription, adequate prescription of statins may limit the disease burden of NAFLD. FUNDING: ZonMw, KWF, NWO, SLO, DGXII, RIDE, National and regional government, Erasmus MC and Erasmus University.

Recapitulating infection, thermal sensitivity and antiviral treatment of seasonal coronaviruses in human airway organoids.

BACKGROUND: Human seasonal coronaviruses usually cause mild upper-respiratory tract infection, but severe complications can occur in specific populations. Research into seasonal coronaviruses is limited and robust experimental models are largely lacking. This study aims to establish human airway organoids (hAOs)-based systems for seasonal coronavirus infection and to demonstrate their applications in studying virus-host interactions and therapeutic development. METHODS: The infections of seasonal coronaviruses 229E, OC43 and NL63 in 3D cultured hAOs with undifferentiated or differentiated phenotypes were tested. The kinetics of virus replication and production was profiled at 33 °C and 37 °C. Genome-wide transcriptome analysis by RNA sequencing was performed in hAOs under various conditions. The antiviral activity of molnupiravir and remdesivir, two approved medications for treating COVID19, was tested. FINDINGS: HAOs efficiently support the replication and infectious virus production of seasonal coronaviruses 229E, OC43 and NL63. Interestingly, seasonal coronaviruses replicate much more efficiently at 33 °C compared to 37 °C, resulting in over 10-fold higher levels of viral replication. Genome-wide transcriptomic analyses revealed distinct patterns of infection-triggered host responses at 33 °C compared to 37 °C temperature. Treatment of molnupiravir and remdesivir dose-dependently inhibited the replication of 229E, OC43 and NL63 in hAOs. INTERPRETATION: HAOs are capable of modeling 229E, OC43 and NL63 infections. The intriguing finding that lower temperature resembling that in the upper respiratory tract favors viral replication may help to better understand the pathogenesis and transmissibility of seasonal coronaviruses. HAOs-based innovative models shall facilitate the research and therapeutic development against seasonal coronavirus infections. FUNDING: This research is supported by funding of a VIDI grant (No. 91719300) from the Netherlands Organization for Scientific Research and the Dutch Cancer Society Young Investigator Grant (10140) to Q.P., and the ZonMw COVID project (114025011) from the Netherlands Organization for Health Research and Development to R.R.

Repurposing dyphylline as a pan-coronavirus antiviral therapy.

Background: In the last two decades, the world has witnessed the emergence of zoonotic corona viruses (CoVs), which cause mild to severe respiratory diseases in humans. Human coronaviruses (HCoVs), mainly from the alpha-CoV and beta-CoV genera, have evolved to be highly pathogenic, such as SARS-CoV-2 causing the COVID-19 pandemic. These coronaviruses carry functional enzymes necessary for the virus life cycle, which represent attractive antiviral targets. Methods & Results: We aimed to therapeutically target the main protease (Mpro) of HCoV-NL63 and HCoV-229E (from alpha-CoV genus) and HCoV-OC43 and SARS-CoV-2 (from beta-CoV genus). Through virtual screening, we identified an FDA-approved drug dyphylline, a xanthine derivate, that binds to the catalytic dyad residues; histidine and cystine of the Mpro structures. Importantly, dyphylline dose-dependently inhibited the viral replication in cell culture models infected with the viruses. Conclusion: Our findings support the repurposing of dyphylline as a pan-coronavirus antiviral agent.

Differing pan-coronavirus antiviral potency of boceprevir and GC376 in vitro despite discordant molecular docking predictions.

Given the structural similarities of the viral enzymes of different coronaviruses (CoVs), we investigated the potency of the anti-SARS-CoV-2 agents boceprevir and GC376 for counteracting seasonal coronavirus infections. In contrast to previous findings that both boceprevir and GC376 are potent inhibitors of the main protease (Mpro) of SARS-CoV-2, we found that GC376 is much more effective than boceprevir in inhibiting SARS-CoV-2 and three seasonal CoVs (NL63, 229E, and OC43) in cell culture models. However, these results are discordant with a molecular docking analysis that suggested comparable affinity of boceprevir and GC376 for the different Mpro enzymes of the four CoVs. Collectively, our results support future development of GC376 but not boceprevir (although it is an FDA-approved antiviral medication) as a pan-coronavirus antiviral agent. Furthermore, we caution against overinterpretation of in silico data when developing antiviral therapies.

Recapitulating hepatitis E virus-host interactions and facilitating antiviral drug discovery in human liver-derived organoids.

Hepatotropic viruses naturally have narrow host and tissue tropisms, challenging the development of robust experimental models. The advent of organoid technology provides a unique opportunity for moving the field forward. Here, we demonstrate that three-dimensional cultured organoids from fetal and adult human liver with cholangiocyte or hepatocyte phenotype support hepatitis E virus (HEV) replication. Inoculation with infectious HEV particles demonstrates that human liver­derived organoids support the full life cycle of HEV infection. By directing organoids toward polarized monolayers in a transwell system, we observed predominantly apical secretion of HEV particles. Genome-wide transcriptomic and tRNAome analyses revealed robust host responses triggered by viral replication. Drug screening in organoids identified brequinar and homoharringtonine as potent HEV inhibitors, which are also effective against the ribavirin resistance variant harboring G1634R mutation. Thus, successful recapitulation of HEV infection in liver-derived organoids shall facilitate the study of virus-host interactions and development of antiviral therapies.

Evaluation of AXIN1 and AXIN2 as targets of tankyrase inhibition in hepatocellular carcinoma cell lines.

AXIN1 mutations are observed in 8-10% of hepatocellular carcinomas (HCCs) and originally were considered to support tumor growth by aberrantly enhancing ß-catenin signaling. This view has however been challenged by reports showing neither a clear nuclear ß-catenin accumulation nor clearly enhanced expression of ß-catenin target genes. Here, using nine HCC lines, we show that AXIN1 mutation or siRNA mediated knockdown contributes to enhanced ß-catenin signaling in all AXIN1-mutant and non-mutant lines, also confirmed by reduced signaling in AXIN1-repaired SNU449 cells. Both AXIN1 and AXIN2 work synergistically to control ß-catenin signaling. While in the AXIN1-mutant lines, AXIN2 is solely responsible for keeping signaling in check, in the non-mutant lines both AXIN proteins contribute to ß-catenin regulation to varying levels. The AXIN proteins have gained substantial interest in cancer research for a second reason. Their activity in the ß-catenin destruction complex can be increased by tankyrase inhibitors, which thus may serve as a therapeutic option to reduce the growth of ß-catenin-dependent cancers. At concentrations that inhibit tankyrase activity, some lines (e.g. HepG2, SNU398) were clearly affected in colony formation, but in most cases apparently independent from effects on ß-catenin signaling. Overall, our analyses show that AXIN1 inactivation leads to enhanced ß-catenin signaling in HCC cell lines, questioning the strong statements that have been made in this regard. Enhancing AXIN activity by tankyrase monotherapy provides however no effective treatment to affect their growth exclusively through reducing ß-catenin signaling.

Differentially Methylated Regions in Desmoid-Type Fibromatosis: A Comparison Between CTNNB1 S45F and T41A Tumors.

INTRODUCTION: The majority of desmoid-type fibromatosis (DTF) tumors harbor a ß-catenin mutation, affecting specific codons in CTNNB1 exon 3. S45F tumors are reported to have a higher chance of recurrence after surgery and more resistance to systemic treatments compared to wild-type (WT) and T41A tumors. The aim of this pilot study was to examine the genome-wide DNA methylation profiles of S45F and T41A mutated DTF, to explain the observed differences in clinical behavior between these DTF subtypes. MATERIAL AND METHODS: Genome-wide analysis of DNA methylation was performed using MeD-seq on formalin-fixed, paraffin-embedded primary DTF samples harboring a S45F (n = 14) or a T41A (n = 15) mutation. Differentially methylated regions (DMRs) between S45F and T41A DTF were identified and used for a supervised hierarchical cluster analysis. DMRs with a fold-change ≥1.5 were considered to be differentially methylated and differences between S45F and T41A tumors were quantitatively assessed. The effect of DMRs on the expression of associated genes was assessed using an independent mRNA expression dataset. Protein-protein interactions between WT ß-catenin and mutant variants and DNA methyltransferase 1 (DNMT1) were examined by immunoprecipitation experiments. RESULTS: MeD-seq analyses indicated 354 regions that displayed differential methylation. Cluster analysis yielded no distinct clusters based on mutation, sex, tumor site or tumor size. A supervised clustering based on DMRs between small (≤34 mm) and large (>87 mm) DTF distinguished the two groups. Only ten DMRs displayed a fold change of ≥1.5 and six of them were found associated with the following genes: NLRP4, FOXK2, PERM1, CCDC6, NOC4L, and DUX4L6. The effects of DMRs on gene expression yielded a significant difference (p < 0.05) in the expression between S45F and T41A for CCDC6 and FOXK2 but not for all Affymetrix probe-sets used to detect these genes. Immunoprecipitations did not reveal an association of WT ß-catenin or mutant variants with DNMT1. CONCLUSION: This study demonstrated that S45F and T41A DTF tumors did not exhibit gross differences in DNA methylation patterns. This implies that distinct DNA methylation profiles are not the sole determinant for the divergent clinical behavior of these different DTF mutant subtypes.

Commonly observed RNF43 mutations retain functionality in attenuating Wnt/ß-catenin signaling and unlikely confer Wnt-dependency onto colorectal cancers.

Cancer-associated RNF43 mutations lead to activation of ß-catenin signaling through aberrantly increasing Wnt-receptor levels at the membrane. Importantly, inactivating RNF43 mutations have been suggested to render cancer cells sensitive to Wnt-based therapeutics. However, the extent to which RNF43 mutations lead to impaired regulation of Wnt/ß-catenin signaling has been poorly investigated. Here, we observed that tumors with a functional mismatch repair system show a predominant 5'-location of truncating RNF43 mutations, suggesting C-terminal truncations such as the most commonly reported p.G659fs mutation, do not affect ß-catenin signaling. In accordance, expressing C-terminal truncation mutants and wild-type RNF43, showed equal effects on ß-catenin signaling, Wnt-receptor turnover, and DVL-binding. We confirmed these observations at endogenous levels by CRISPR-Cas9-mediated knockout of G659fs RNF43 expression in KM12 cells and generating comparable mutations in HEK293T cells. We could not confirm previous reports linking RNF43 to p53 and E-cadherin breakdown. Our data also suggest that only colorectal cancer cells harboring N-terminal mutations of RNF43 convey Wnt-dependency onto the tumor cells. Results of this study have potentially important clinical implications indicating that Wnt-based therapeutics should be applied cautiously in cancer patients harboring RNF43 mutations.

Oncogenic Mutations in Armadillo Repeats 5 and 6 of ß-Catenin Reduce Binding to APC, Increasing Signaling and Transcription of Target Genes.

BACKGROUND & AIMS: The ß-catenin signaling pathway is one of the most commonly deregulated pathways in cancer cells. Amino acid substitutions within armadillo repeats 5 and 6 (K335, W383, and N387) of ß-catenin are found in several tumor types, including liver tumors. We investigated the mechanisms by which these substitutions increase signaling and the effects on liver carcinogenesis in mice. METHODS: Plasmids encoding tagged full-length ß-catenin (CTNNB1) or ß-catenin with the K335I or N387K substitutions, along with MET, were injected into tails of FVB/N mice. Tumor growth was monitored, and livers were collected and analyzed by histology, immunohistochemistry, and quantitative reverse-transcription polymerase chain reaction. Tagged full-length and mutant forms of ß-catenin were expressed in HEK293, HCT116, and SNU449 cells, which were analyzed by immunoblots and immunoprecipitation. A panel of ß-catenin variants and cell lines with knock-in mutations were analyzed for differences in N-terminal phosphorylation, half-life, and association with other proteins in the signaling pathway. RESULTS: Mice injected with plasmids encoding K335I or N387K ß-catenin and MET developed larger, more advanced tumors than mice injected with plasmids encoding WT ß-catenin and MET. K335I and N387K ß-catenin bound APC with lower affinity than WT ß-catenin but still interacted with scaffold protein AXIN1 and in the nucleus with TCF7L2. This interaction resulted in increased transcription of genes regulated by ß-catenin. Studies of protein structures supported the observed changes in relative binding affinities. CONCLUSION: Expression of ß-catenin with mutations in armadillo repeats 5 and 6, along with MET, promotes formation of liver tumors in mice. In contrast to N-terminal mutations in ß-catenin that directly impair its phosphorylation by GSK3 or binding to BTRC, the K335I or N387K substitutions increase signaling via reduced binding to APC. However, these mutant forms of ß-catenin still interact with the TCF family of transcription factors in the nucleus. These findings show how these amino acid substitutions increase ß-catenin signaling in cancer cells.

Oncogenic STRAP Supports Hepatocellular Carcinoma Growth by Enhancing Wnt/ß-Catenin Signaling.

Aberrant activation of Wnt/ß-catenin signaling plays a key role in the onset and development of hepatocellular carcinomas (HCC), with about half of them acquiring mutations in either CTNNB1 or AXIN1. The serine/threonine kinase receptor-associated protein (STRAP), a scaffold protein, was recently shown to facilitate the aberrant activation of Wnt/ß-catenin signaling in colorectal cancers. However, the function of STRAP in HCC remains completely unknown. Here, increased levels of STRAP were observed in human and mouse HCCs. RNA sequencing of STRAP knockout clones generated by gene editing of Huh6 and Huh7 HCC cells revealed a significant reduction in expression of various metabolic and cell-cycle-related transcripts, in line with their general slower growth observed during culture. Importantly, Wnt/ß-catenin signaling was impaired in all STRAP knockout/down cell lines tested, regardless of the underlying CTNNB1 or AXIN1 mutation. In accordance with ß-catenin's role in (cancer) stem cell maintenance, the expressions of various stem cell markers, such as AXIN2 and LGR5, were reduced and concomitantly differentiation-associated genes were increased. Together, these results show that the increased STRAP protein levels observed in HCC provide growth advantage among others by enhancing Wnt/ß-catenin signaling. These observations also identify STRAP as a new player in regulating ß-catenin signaling in hepatocellular cancers. IMPLICATIONS: Elevated STRAP levels in hepatocellular cancers provide a growth advantage by enhancing Wnt/ß-catenin signaling.

Identification of SLC41A3 as a novel player in magnesium homeostasis.

Regulation of the body Mg(2+) balance takes place in the distal convoluted tubule (DCT), where transcellular reabsorption determines the final urinary Mg(2+) excretion. The basolateral Mg(2+) extrusion mechanism in the DCT is still unknown, but recent findings suggest that SLC41 proteins contribute to Mg(2+) extrusion. The aim of this study was, therefore, to characterize the functional role of SLC41A3 in Mg(2+) homeostasis using the Slc41a3 knockout (Slc41a3(-/-)) mouse. By quantitative PCR analysis it was shown that Slc41a3 is the only SLC41 isoform with enriched expression in the DCT. Interestingly, serum and urine electrolyte determinations demonstrated that Slc41a3(-/-) mice suffer from hypomagnesemia. The intestinal Mg(2+) absorption capacity was measured using the stable (25)Mg(2+) isotope in mice fed a low Mg(2+) diet. (25)Mg(2+) uptake was similar in wildtype (Slc41a3(+/+)) and Slc41a3(-/-) mice, although Slc41a3(-/-) animals exhibited increased intestinal mRNA expression of Mg(2+) transporters Trpm6 and Slc41a1. Remarkably, some of the Slc41a3(-/-) mice developed severe unilateral hydronephrosis. In conclusion, SLC41A3 was established as a new factor for Mg(2+) handling.

Urinary ß-galactosidase stimulates Ca2+ transport by stabilizing TRPV5 at the plasma membrane.

Transcellular Ca(2+)transport in the late distal convoluted tubule and connecting tubule (DCT2/CNT) of the kidney is a finely controlled process mediated by the transient receptor potential vanilloid type 5 (TRPV5) channel. A complex-type-N-glycan bound at the extracellular residue Asn358 of TRPV5 through post-translational glycosylation has been postulated to regulate the activity of TRPV5 channels. Using in vitro Ca(2+)transport assays, immunoblot analysis, immunohistochemistry, patch clamp electrophysiology and total internal reflection fluorescence microscopy, it is demonstrated that the glycosidase ß-galactosidase (ß-gal), an enzyme that hydrolyzes galactose, stimulates TRPV5 channel activity. However, the activity of the non-glycosylated TRPV(N358Q)mutant was not altered in the presence of ß-gal, showing that the stimulation is dependent on the presence of the TRPV5N-glycan. In addition, ß-gal was found to stimulate transcellular Ca(2+)transport in isolated mouse primary DCT2/CNT cells. ß-gal expression was detected in the apical membrane of the proximal tubules, and the protein was found in mouse urine. In summary, ß-gal is present in the pro-urine from where it is thought to stimulate TRPV5 activity.

Shedding of klotho by ADAMs in the kidney.

The anti-aging gene klotho plays an important role in Ca(2+) and phosphate homeostasis. Membrane-bound klotho is an essential coreceptor for fibroblast growth factor-23 and can be cleaved by proteases, including a disintegrin and metalloproteinase (ADAM)10 and ADAM17. Cleavage of klotho occurs at a site directly above the plasma membrane (α-cut) or between the KL1 and KL2 domain (ß-cut), resulting in soluble full-length klotho or KL1 and KL2 fragments, respectively. The aim of the present study was to gain insights into the mechanisms behind klotho cleavage processes in the kidney. Klotho shedding was demonstrated using a Madin-Darby canine kidney cell line stably expressing klotho and human embryonic kidney-293 cells transiently transfected with klotho. Here, we report klotho expression on both the basolateral and apical membrane, with a higher abundance of klotho at the apical membrane and in the apical media. mRNA expression of ADAM17 and klotho were enriched in mouse distal convoluted and connecting tubules. In vitro ADAM/matrix metalloproteinase inhibition by TNF484 resulted in a concentration-dependent inhibition of the α-cut, with a less specific effect on ß-cut shedding. In vivo TNF484 treatment in wild-type mice did not change urinary klotho levels. However, ADAM/matrix metalloproteinase inhibition did increase renal and duodenal mRNA expression of phosphate transporters, whereas serum phosphate levels were significantly decreased. In conclusion, our data show that renal cells preferentially secrete klotho to the apical side and suggest that ADAMs are responsible for α-cut cleavage.