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Vitamin B12 is an essential nutrient acquired via dietary intake. Receptor-mediated endocytosis is a key mechanism in vitamin B12 absorption, cellular uptake, and reabsorption. CD320 is a type I transmembrane protein responsible for cellular uptake of vitamin B12 in peripheral tissues. In this study, we examined segmental distribution and cellular expression of CD320 in mouse kidneys and intestines. We show that CD320 is expressed on the luminal surface in the small intestine and in proximal tubules in the kidney, suggesting that, in addition to its role in vitamin B12 uptake in peripheral tissues, CD320 may participate in vitamin B12 absorption in the small intestine and reabsorption in the kidney. Moreover, we show that an amino acid motif, DSSDE, in the second low-density lipoprotein receptor class A domain of CD320 is a key apical membrane targeting signal in both renal and intestinal epithelial cells. Mutations or deletion of this motif abolish the specific apical membrane expression of CD320 in polarized Madin-Darby canine kidney cells and human colon cancer-derived Caco-2 cells. In short-hairpin RNA-based gene knockdown experiments, we show that the apical membrane targeting of CD320 is mediated by a Rab11a-dependent mechanism. These results extend our knowledge regarding the cell biology of CD320 and its role in vitamin B12 metabolism.

The biological and clinical significance of the p.E88del variant in the transcobalamin receptor, CD320, is unknown. This allele is annotated in ClinVar as likely benign, pathogenic, and of uncertain significance. To determine functional consequence and clinical relevance of this allele, we employed cell culture and genetic association studies. Fibroblasts from sixteen CD320 p.E88del homozygotes exhibited reduced binding and uptake of cobalamin. Complete ascertainment of newborns with transiently elevated C3 (propionylcarnitine) in New York State demonstrated that homozygosity for CD320 p.E88del was over-represented (7/348, p

Atrial natriuretic peptide (ANP) is of major importance in the maintenance of electrolyte balance and normal blood pressure. Reduced plasma ANP levels are associated with the increased risk of cardiovascular disease. Corin is a type II transmembrane serine protease that converts the ANP precursor to mature ANP. Corin deficiency prevents ANP generation and alters electrolyte and body fluid homeostasis. Corin is synthesized as a zymogen that is proteolytically activated on the cell surface. Factors that disrupt corin folding, intracellular trafficking, cell surface expression, and zymogen activation are expected to impair corin function. To date, CORIN variants that reduce corin activity have been identified in hypertensive patients. In addition to the heart, corin expression has been detected in non-cardiac tissues, where corin and ANP participate in diverse physiological processes. In this review, we summarize the current knowledge in corin biosynthesis and post-translational modifications. We also discuss tissue-specific corin expression and function in physiology and disease.

Transmembrane protease serine 2 (TMPRSS2) is a membrane-bound protease expressed in many human epithelial tissues, including the airway and lung. TMPRSS2-mediated cleavage of viral spike protein is a key mechanism in severe acute respiratory syndrome coronavirus 2 activation and host cell entry. To date, the cellular mechanisms that regulate TMPRSS2 activity and cell surface expression are not fully characterized. In this study, we examined two major post-translational events, zymogen activation and N-glycosylation, in human TMPRSS2. In experiments with human embryonic kidney 293, bronchial epithelial 16HBE, and lung alveolar epithelial A549 cells, we found that TMPRSS2 was activated via intracellular autocatalysis and that this process was blocked in the presence of hepatocyte growth factor activator inhibitors 1 and 2. By glycosidase digestion and site-directed mutagenesis, we showed that human TMPRSS2 was N-glycosylated. N-glycosylation at an evolutionarily conserved site in the scavenger receptor cysteine-rich domain was required for calnexin-assisted protein folding in the endoplasmic reticulum and subsequent intracellular trafficking, zymogen activation, and cell surface expression. Moreover, we showed that TMPRSS2 cleaved severe acute respiratory syndrome coronavirus 2 spike protein intracellularly in human embryonic kidney 293 cells. These results provide new insights into the cellular mechanism in regulating TMPRSS2 biosynthesis and function. Our findings should help to understand the role of TMPRSS2 in major respiratory viral diseases.

Atrial natriuretic peptide (ANP) is an important hormone in cardiovascular biology. It is activated by the protease corin. In pregnancy, ANP and corin promote uterine spiral artery remodeling, but the underlying mechanism remains unknown. Here we report an ANP function in uterine decidualization and TNF-related apoptosis-inducing ligand-dependent (TRAIL-dependent) death in spiral arterial smooth muscle cells (SMCs) and endothelial cells (ECs). In ANP- or corin-deficient mice, uterine decidualization markers and TRAIL expression were decreased, whereas in cultured human endometrial stromal cells (HESCs), ANP increased decidualization and TRAIL expression. In uterine spiral arteries from pregnant wild-type mice, SMC and EC loss occurred sequentially before trophoblast invasion. In culture, TRAIL from decidualized HESCs induced apoptosis in uterine SMCs, but not in ECs with low TRAIL receptor expression. Subsequently, cyclophilin B was identified from apoptotic SMCs that upregulated endothelial TRAIL receptor and caused apoptosis in ECs. These results indicate that ANP promotes decidualization and TRAIL expression in endometrial stromal cells, contributing to sequential events in remodeling of spiral arteries, including SMC death and cyclophilin B release, which in turn induces TRAIL receptor expression and apoptosis in ECs.

Transmembrane serine proteases have been implicated in the development and progression of solid and hematological cancers. Human airway trypsin‐like protease 4 (HAT‐L4) is a transmembrane serine protease expressed in epithelial cells and exocrine glands. In the skin, HAT‐L4 is important for normal epidermal barrier function. Here, we report an unexpected finding of ectopic HAT‐L4 expression in neutrophils and monocytes from acute myeloid leukemia (AML) patients. Such expression was not detected in bone marrow cells from normal individuals or patients with chronic myeloid leukemia, acute lymphocytic leukemia and chronic lymphocytic leukemia. In AML patients who underwent chemotherapy, persistent HAT‐L4 expression in bone marrow cells was associated with minimal residual disease and poor prognostic outcomes. In culture, silencing HAT‐L4 expression in AML–derived THP‐1 cells by short hairpin RNAs inhibited matrix metalloproteinase‐2 activation and Matrigel invasion. In mouse xenograft models, inhibition of HAT‐L4 expression reduced the proliferation and growth of THP‐1 cell–derived tumors. Our results indicate that ectopic HAT‐L4 expression is a pathological mechanism in AML and that HAT‐L4 may be used as a cell surface marker for AML blast detection and targeting.

Selective protein distribution on distinct plasma membranes is important for epithelial cell function. To date, how proteins are directed to specific epithelial cell surface is not fully understood. Here we report a conserved DSSDE motif in LDL-receptor (LDLR) modules of corin (a transmembrane serine protease) and CD320 (a receptor for vitamin B12 uptake), which regulates apical membrane targeting in renal epithelial cells. Altering this motif prevents specific apical corin and CD320 expression in polarized Madin–Darby canine kidney (MDCK) cells. Mechanistic studies indicate that this DSSDE motif participates in a Rab11a-dependent mechanism that specifies apical sorting. In MDCK cells, inhibition of Rab11a, but not Rab11b, expression leads to corin and CD320 expression on both apical and basolateral membranes. Together, our results reveal a novel molecular recognition mechanism that regulates LDLR module-containing proteins in their specific apical expression in polarized renal epithelial cells.

Spiral artery remodeling is an important physiological process in the pregnant uterus which increases blood flow to the fetus. Impaired spiral artery remodeling contributes to preeclampsia, a major disease in pregnancy. Corin, a transmembrane serine protease, is up-regulated in the pregnant uterus to promote spiral artery remodeling. To date, the mechanism underlying uterine corin up-regulation remains unknown. Here we show that Kruppel-like factor (KLF) 17 is a key transcription factor for uterine corin expression in pregnancy. In cultured human uterine endometrial cells, KLF17 binds to the CORIN promoter and enhances the promoter activity. Disruption of the KLF17 gene in the endometrial cells abolishes CORIN expression. In mice, Klf17 is up-regulated in the pregnant uterus. Klf17 deficiency prevents uterine Corin expression in pregnancy. Moreover, Klf17-deficient mice have poorly remodeled uterine spiral arteries and develop gestational hypertension and proteinuria. Together, our results reveal an important function of KLF17 in regulating Corin expression and uterine physiology in pregnancy.

Corin is a transmembrane serine protease that activates atrial natriuretic peptide, a cardiac hormone essential for normal blood pressure. Corin is synthesized as a zymogen and activated on the cell surface. In previous studies, we identified a CORIN variant allele with an adenine insertion in the 5'-end of the coding region in ∼5% of hypertensive individuals in a Chinese population. The protein, named insA, encoded by the CORIN variant allele has a shortened cytoplasmic tail and reduced atrial natriuretic peptide processing activity. It remains unknown how a shortened cytoplasmic tail impairs corin function. In this study, we expressed a series of corin mutants with different N-terminal sequences and analyzed them by Western blotting, flow cytometry, protein chase, and immunostaining. Our results revealed that a Gly-Asn sequence after the initiating Met at the newly generated N-terminus was responsible for delaying corin trafficking in the Golgi. Deletion of the N-terminal Gly and Asn residues increased the intracellular trafficking, cell surface expression, and activation cleavage of the insA variant. These results help to explain how the CORIN variant allele impairs corin structure and function as an underlying mechanism in hypertension.

Type II transmembrane serine proteases (TTSPs) are a group of enzymes participating in diverse biological processes. Some members of the TTSP family are implicated in viral infection. TMPRSS11A is a TTSP expressed on the surface of airway epithelial cells, which has been shown to cleave and activate spike proteins of the severe acute respiratory syndrome (SARS) and the Middle East respiratory syndrome coronaviruses (CoVs). In this study, we examined the mechanism underlying the activation cleavage of TMPRSS11A that converts the one-chain zymogen to a two-chain enzyme. By expression in human embryonic kidney 293, esophageal EC9706, and lung epithelial A549 and 16HBE cells, Western blotting, and site-directed mutagenesis, we found that the activation cleavage of human TMPRSS11A was mediated by autocatalysis. Moreover, we found that TMPRSS11A activation cleavage occurred before the protein reached the cell surface, as indicated by studies with trypsin digestion to remove cell surface proteins, treatment with cell organelle-disturbing agents to block intracellular protein trafficking, and analysis of a soluble form of TMPRSS11A without the transmembrane domain. We also showed that TMPRSS11A was able to cleave the SARS-CoV-2 spike protein. These results reveal an intracellular autocleavage mechanism in TMPRSS11A zymogen activation, which differs from the extracellular zymogen activation reported in other TTSPs. These findings provide new insights into the diverse mechanisms in regulating TTSP activation.

The group A scavenger receptor cysteine-rich (SRCR) domain is a conserved module present in numerous proteins involved in diverse biological processes. Hepsin, a hepatic protease implicated in many cancers, consists of a cytoplasmic tail, a transmembrane domain and an extracellular regions with a group A SRCR domain and a serine protease domain. Like in many SRCR-containing proteins, the SRCR domain in hepsin has an N-glycosylation site, but its functional significance is unknown. In this study, we confirmed N-glycosylation at Asn112 in hepsin by glycosidase digestion and site-directed mutagenesis in human hepatoma cells. In Western blotting, fluorogenic substrate assay, flow cytometry, and protein-chase experiments, we found that Asn112 to Gln (N112Q) mutation inhibited hepsin intracellular trafficking, cell surface expression, and zymogen activation. By immunofluorescent staining, we found that the N112Q mutant was more abundant than wild-type hepsin in the endoplasmic reticulum (ER). Further co-immunoprecipitation studies indicated increased binding of the N112Q mutant to calnexin and binding-immunoglobulin protein (BiP), two ER chaperones. Our results indicate that the N-glycan in the SRCR domain of hepsin promotes intracellular trafficking and cell surface expression, possibly by a calnexin-dependent mechanism in facilitating ER exiting.

Membrane-bound proteases are essential for epidermal integrity. Human airway trypsin-like protease 4 (HAT-L4) is a type II transmembrane serine protease. Currently, its biochemical property, cellular distribution and physiological function remain unknown. Here we examined HAT-L4 expression and function in vitro and in vivo. In Western analysis, HAT-L4 expressed in transfected CHO cells appeared as a 48-kDa protein. Flow cytometry confirmed HAT-L4 expression on the cell surface with the expected membrane topology. RT-PCR and immunostaining experiments indicated that HAT-L4 was expressed in epithelial cells and exocrine glands in tissues including skin, esophagus, trachea, tongue, eye, bladder, testis and uterus. In the skin, HAT-L4 expression was abundant in keratinocytes and sebaceous glands. We generated HAT-L4 knockout mice by disrupting the Tmprss11f gene encoding HAT-L4. HAT-L4 knockout mice were viable and fertile. No defects were found in HAT-L4 knockout mice in hair growth, wound healing, water repulsion and body temperature regulation. Compared with wild-type controls, HAT-L4-deficient newborn mice had greater body fluid loss and higher mortality in a trans-epidermal body fluid loss test. In metabolic studies, HAT-L4-deficient adult mice drank water more frequently than wild-type controls did. These results indicate that HAT-L4 is important in epidermal barrier function to prevent body fluid loss.

Pre-eclampsia (PE) is a chronic inflammatory disease in pregnancy, which is associated with enhanced blood coagulation and high thrombotic risk. To date, the mechanisms underlying such an association are not fully understood. Previous studies reported high levels of plasma deoxyribonucleic acid (DNA) in PE women, but the cellular source of the circulation DNA remains unknown. In this study, we tested the hypothesis that activated neutrophils undergoing cell death, also called NETosis, may be responsible for the elevated plasma DNA levels in PE women. We analysed plasma samples from non-pregnant, normal pregnant and PE women and found high levels of double-stranded DNA, myeloperoxidase (an abundant neutrophil granular enzyme) and histones (the major nucleosome proteins) in PE-derived samples, indicating increased NETosis in the maternal circulation. The high plasma DNA levels positively correlated with enhanced blood coagulation in PE women. When isolated neutrophils from normal individuals were incubated with PE-derived plasma, an elevated NETosis-stimulating activity was detected. Further experiments showed that endothelial micro-particles, but not soluble proteins, in the plasma were primarily responsible for the NETosis-stimulating activity in PE women. These results indicate that circulating micro-particles from damaged maternal endothelium are a potent stimulator for neutrophil activation and NETosis in PE women. Given the pro-coagulant and pro-thrombotic nature of granular and nuclear contents from neutrophils, enhanced systemic NETosis may represent an important mechanism underlying the hyper-coagulability and increased thrombotic risk in PE.

Corin has been studied extensively within the vascular system and is known to regulate blood pressure. We have shown that corin is one of the most highly upregulated genes during osteogenic differentiation of human adipose‐derived stem cells (hASCs). This study tested the hypothesis that, through modulation of angiogenic signalling pathways, corin is a critical regulator of osteogenic differentiation and endochondral ossification. In vitro, corin expression in hASC was suppressed via siRNA knockdown and vascular endothelial growth factor A (VEGF‐A) expression was quantified via reverse transcription polymerase chain reaction. In vivo, a murine corin knockout model (female, 10 weeks) was used to determine the effect of corin deficiency on long bone development. Wild‐type and corin knockout long bones were compared via haematoxylin and eosin staining to assess tissue characteristics and cellular organization, three‐point bending to assess mechanical characteristics, and immunohistochemistry to visualize VEGF‐A expression patterns. Corin knockdown significantly (p < 0.05) increased VEGF‐A mRNA expression during osteogenic differentiation. In vivo, corin knockout reduced tibial growth plate thickness (p < 0.01) and severely diminished the hypertrophic region. Corin knockout femurs had significantly increased stiffness (p < 0.01) and maximum loads (p < 0.01) but reduced postyield deflections (p < 0.01). In corin knockout mice, VEGF‐A expression was increased near the growth plate but was reduced throughout the tibial shaft and distal head of the tibiae. This is the first study to show that corin is a key regulator of bone development by modulation of VEGF‐A expression. Further elucidation of this mechanism will aid in the development of optimized bone tissue engineering and regenerative medicine therapies.

Over the past decade, cell therapies have provided promising strategies for the treatment of ischaemic cardiomyopathy. Particularly, the beneficial effects of stem cells, including bone marrow stem cells (BMSCs), endothelial progenitor cells (EPCs), mesenchymal stem cells (MSCs), embryonic stem cells (ESCs), and induced pluripotent stem cells (iPSCs), have been demonstrated by substantial preclinical and clinical studies. Nevertheless stem cell therapy is not always safe and effective. Hence, there is an urgent need for alternative sources of cells to promote cardiac regeneration. Human villous trophoblasts (HVTs) play key roles in embryonic implantation and placentation. In this study, we show that HVTs can promote tube formation of human umbilical vein endothelial cells (HUVECs) on Matrigel and enhance the resistance of neonatal rat cardiomyocytes (NRCMs) to oxidative stress in vitro. Delivery of HVTs to ischaemic area of heart preserved cardiac function and reduced fibrosis in a mouse model of acute myocardial infarction (AMI). Histological analysis revealed that transplantation of HVTs promoted angiogenesis in AMI mouse hearts. In addition, our data indicate that HVTs exert their therapeutic benefit through paracrine mechanisms. Meanwhile, injection of HVTs to mouse hearts did not elicit severe immune response. Taken together, our study demonstrates HVT may be used as a source for cell therapy or a tool to study cell‐derived soluble factors for AMI treatment.

Hepsin is a transmembrane serine protease implicated in many biological processes, including hepatocyte growth, urinary protein secretion, auditory nerve development, and cancer metastasis. Zymogen activation is critical for hepsin function. To date, how hepsin is activated and regulated in cells remains an enigma. In this study, we conducted site-directed mutagenesis, cell expression, plasma membrane protein labeling, trypsin digestion, Western blotting, and flow cytometry experiments in human hepatoma HepG2 cells, where hepsin was originally discovered, and SMMC-7721 cells. Our results show that hepsin is activated by autocatalysis on the cell surface but not intracellularly. Moreover, we show that hepsin undergoes ectodomain shedding. In the conditioned medium from HepG2 and SMMC-7721 cells, we detected a soluble fragment comprising nearly the entire extracellular region of hepsin. By testing protease inhibitors, gene knockdown, and site-directed mutagenesis, we identified calpain-1 as a primary protease that acted extracellularly to cleave Tyr52 in the juxtamembrane space of hepsin. These results provide new insights into the biochemical and cellular mechanisms that regulate hepsin expression and activity.

Factor VII (FVII) is a key serine protease in blood coagulation. N-glycosylation in FVII has been shown to be critical for protein secretion. To date, however, the underlying biochemical mechanism remains unclear. Recently, we found that N-glycans in the transmembrane serine protease corin are critical for calnexin-assisted protein folding and extracellular expression. In this study, we tested the hypothesis that N-glycans in the FVII protease domain mediate calnexin-assisted protein folding and that naturally occurring F7 mutations abolishing N-glycosylation impair FVII secretion. We expressed human FVII wild-type (WT) and mutant proteins lacking one or both N-glycosylation sites in HEK293 and HepG2 cells in the presence or absence of a glucosidase inhibitor. FVII expression, secretion and binding to endoplasmic reticulum chaperones were examined by immune staining, co-immunoprecipitation, Western blotting, and ELISA. We found that N-glycosylation at N360 in the protease domain, but not N183 in the pro-peptide domain, of human FVII is required for protein secretion. Elimination of N-glycosylation at N360 impaired calnexin-assisted FVII folding and secretion. Similar results were observed in WT FVII when N-glycan-calnexin interaction was blocked by glucosidase inhibition. Naturally occurring F7 mutations abolishing N-glycosylation at N360 reduced FVII secretion in HEK293 and HepG2 cells. These results indicate that N-glycans in the FVII protease domain mediate calnexin-assisted protein folding and subsequent extracellular expression. Naturally occurring F7 mutations abolishing N-glycosylation in FVII may impair this mechanism, thereby reducing FVII levels in patients.

Sweating is a basic skin function in body temperature control. In sweat glands, salt excretion and reabsorption are regulated to avoid electrolyte imbalance. To date, the mechanism underlying such regulation is not fully understood. Corin is a transmembrane protease that activates atrial natriuretic peptide (ANP), a cardiac hormone essential for normal blood volume and pressure. Here, we report an unexpected role of corin in sweat glands to promote sweat and salt excretion in regulating electrolyte homeostasis. In human and mouse eccrine sweat glands, corin and ANP are expressed in the luminal epithelial cells. In corin-deficient mice on normal- and high-salt diets, sweat and salt excretion is reduced. This phenotype is associated with enhanced epithelial sodium channel (ENaC) activity that mediates Na+ and water reabsorption. Treatment of amiloride, an ENaC inhibitor, normalizes sweat and salt excretion in corin-deficient mice. Moreover, treatment of aldosterone decreases sweat and salt excretion in wild-type (WT), but not corin-deficient, mice. These results reveal an important regulatory function of corin in eccrine sweat glands to promote sweat and salt excretion., Sweating is a basic skin function in body temperature control, and salt excretion and reabsorption in sweat glands are essential for salt-water balance. This study identifies corin, a transmembrane protease that activates atrial natriuretic peptide, as a key enzyme in regulating salt excretion in the skin.

Corin is a membrane-bound protease essential for activating natriuretic peptides and regulating blood pressure. Human corin has 19 predicted N-glycosylation sites in its extracellular domains. It has been shown that N-glycans are required for corin cell surface expression and zymogen activation. It remains unknown, however, how N-glycans at different sites may regulate corin biosynthesis and processing. In this study, we examined corin mutants, in which each of the 19 predicted N-glycosylation sites was mutated individually. By Western analysis of corin proteins in cell lysate and conditioned medium from transfected HEK293 cells and HL-1 cardiomyocytes, we found that N-glycosylation at Asn-80 inhibited corin shedding in the juxtamembrane domain. Similarly, N-glycosylation at Asn-231 protected corin from autocleavage in the frizzled-1 domain. Moreover, N-glycosylation at Asn-697 in the scavenger receptor domain and at Asn-1022 in the protease domain is important for corin cell surface targeting and zymogen activation. We also found that the location of the N-glycosylation site in the protease domain was not critical. N-Glycosylation at Asn-1022 may be switched to different sites to promote corin zymogen activation. Together, our results show that N-glycans at different sites may play distinct roles in regulating the cell membrane targeting, zymogen activation, and ectodomain shedding of corin.

Objective— Thrombophilia is a major complication in preeclampsia, a disease associated with placental hypoxia and trophoblast inflammation. Preeclampsia women are known to have increased circulating microparticles that are procoagulant, but the underlying mechanisms remain unclear. In this study, we sought to understand the mechanism connecting placental hypoxia, circulating microparticles, and thrombophilia. Approach and Results— We analyzed protein markers on plasma microparticles from preeclampsia women and found that the increased circulating microparticles were mostly from endothelial cells. In proteomic studies, we identified HMGB1 (high-mobility group box 1), a proinflammatory protein, as a key factor from hypoxic trophoblasts in stimulating microparticle production in human umbilical vein endothelial cells. Immunodepletion or inhibition of HMGB1 in the conditioned medium from hypoxic human trophoblasts abolished the endothelial microparticle-stimulating activity. Conversely, recombinant HMGB1 stimulated microparticle production in cultured human umbilical vein endothelial cells. The microparticles from recombinant HMGB1–stimulated human umbilical vein endothelial cells promoted blood coagulation and neutrophil activation in vitro. Injection of recombinant HMGB1 in pregnant mice increased plasma endothelial microparticles and promoted blood coagulation. In preeclampsia women, elevated placental HMGB1 expression was detected and high levels of plasma HMGB1 correlated with increased plasma endothelial microparticles. Conclusions— Our results indicate that placental hypoxia-induced HMGB1 expression and release from trophoblasts are important mechanism underlying increased circulating endothelial microparticles and thrombophilia in preeclampsia.

Corin is a serine protease that is important for the regulation of blood pressure and water balance. Corin was initially discovered in the heart, however, it has also been detected in kidney cells, though its function in the kidneys is unclear. To further investigate the function of corin in the kidney, the present study analyzed the levels of corin in urine and blood samples collected from normal individuals and patients with primary proteinuric diseases. The associations between the levels of corin, and the cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) were then assessed. The results demonstrated that corin was detectable in the urine and plasma following an enzyme-linked immunosorbent assay; the level of corin in the urine was associated with the level of urinary β2-microglobulin (P=0.01), which was indicative of renal tubular injury. When compared with normal individuals, the levels of urinary corin in proteinuric patients were markedly increased (P=0.02), and were also associated with IL-1β (P=0.03). This correlation between corin and IL-1β was confirmed in vitro using 293 cells. As the IL-1β concentrations increased (0, 0.1, 1, 10 ng/ml), an elevation in the level of corin was observed in the culture medium (P0.05). In addition, when TNF-α reached 10 ng/ml, the level of corin in the medium increased significantly when compared with the control group (0 ng/ml; P=0.02), however, no significant difference in corin levels was detected in the cell lysate. The results suggest that the cytokines IL-1β and TNF-α may increase urinary corin in patients with primary proteinuric kidney diseases. Cytokines may accelerate corin shedding from the cell membrane of renal tubule epithelial cells. These findings indicate that corin may be associated with kidney inflammation and injury.

Corin is a serine protease that activates atrial natriuretic peptide (ANP). CORIN gene variants have been reported in patients with hypertension. To date, however, the prevalence of CORIN variants in hypertensive patients remains unknown. To understand the prevalence and functional significance of CORIN variants in hypertension, we sequenced CORIN exons in 300 normal and 401 hypertensive individuals in a Chinese population and identified nine nonsynonymous variants, of which eight were not characterized previously. Among them, variants c.131A > G (p.Tyr13Cys), c.376G > T (p.Asp95Tyr), c.1094T > G (p.Leu334Trp), and c.1667G > A (p.Arg525His) occurred similarly in both normal and hypertensive individuals. Variants c1139G > A (p.Arg349His), c.2689C > T (p.Pro866Ser), and c.2864C > T (p.Thr924Met) were found once each in hypertensive individuals. Variant c.1683G > T (p.Arg530Ser) occurred preferentially in hypertensive individuals [10/401 (2.5%) vs. 1/300 (0.3%) in normal individuals; P = 0.023], which was confirmed in another independent cohort [9/368 (2.44%) in hypertensive and 2/377 (0.53%) in normal individuals; P = 0.033]. In biochemical and cell-based functional studies, variants p.Arg530Ser and p.Thr924Met, but not p.Tyr13Cys, p.Asp95Tyr, p.Leu334Trp, p.Arg349His, p.Arg525His, and p.Pro866Ser, exhibited reduced pro-ANP processing activity, which was caused by endoplasmic reticulum retention and poor zymogen activation, respectively. These results indicate that genetic variants impairing corin function are not uncommon in general populations and that such variants may be an important contributing factor in hypertension.

Membrane-bound proteases are essential for epidermal integrity. Human airway trypsin-like protease 4 (HAT-L4) is a type II transmembrane serine protease. Currently, its biochemical property, cellular distribution and physiological function remain unknown. Here we examined HAT-L4 expression and function in vitro and in vivo. In Western analysis, HAT-L4 expressed in transfected CHO cells appeared as a 48-kDa protein. Flow cytometry confirmed HAT-L4 expression on the cell surface with the expected membrane topology. RT-PCR and immunostaining experiments indicated that HAT-L4 was expressed in epithelial cells and exocrine glands in tissues including skin, esophagus, trachea, tongue, eye, bladder, testis and uterus. In the skin, HAT-L4 expression was abundant in keratinocytes and sebaceous glands. We generated HAT-L4 knockout mice by disrupting the Tmprss11f gene encoding HAT-L4. HAT-L4 knockout mice were viable and fertile. No defects were found in HAT-L4 knockout mice in hair growth, wound healing, water repulsion and body temperature regulation. Compared with wild-type controls, HAT-L4-deficient newborn mice had greater body fluid loss and higher mortality in a trans-epidermal body fluid loss test. In metabolic studies, HAT-L4-deficient adult mice drank water more frequently than wild-type controls did. These results indicate that HAT-L4 is important in epidermal barrier function to prevent body fluid loss.

Bone marrow-derived endothelial progenitor cells (EPCs) are being tested as a therapy to treat a variety of ischemic diseases. Poor homing to targeted tissues is one of the major factors limiting the therapeutic efficacy of EPCs. Here, we show that human cord blood-derived EPCs expressed little sialyl Lewis X (sLe(x)) antigen that is necessary for selectin-mediated cell-cell interactions. Expression of α1,3-fucosyltransferase VI (FucT VI) in the EPCs enhanced sLe(x) synthesis, E- and P-selectin-binding, and EPC adhesion to tumor necrosis factor-α-stimulated human umbilical vein endothelial cells in culture. In a mouse model of hind limb ischemia, in which EPCs were injected intravenously, FucT VI expression increased EPC homing, neovascularization, and blood flow in ischemic muscles. In another mouse model of femoral fracture, FucT VI-expressing EPCs were more efficient than control EPCs in targeting to peri-fracture tissues to enhance angiogenesis, blood flow and bone repair. These results indicate that fucosylated EPCs may be used to as an improved cellular source to treat ischemic diseases.

Mechanisms underlying the vein development remain largely unknown. Tie2 signaling mediates endothelial cell (EC) survival and vascular maturation and its activating mutations are linked to venous malformations. Here we show that vein formation are disrupted in mouse skin and mesentery when Tie2 signals are diminished by targeted deletion of Tek either ubiquitously or specifically in embryonic ECs. Postnatal Tie2 attenuation resulted in the degeneration of newly formed veins followed by the formation of haemangioma-like vascular tufts in retina and venous tortuosity. Mechanistically, Tie2 insufficiency compromised venous EC identity, as indicated by a significant decrease of COUP-TFII protein level, a key regulator in venogenesis. Consistently, angiopoietin-1 stimulation increased COUP-TFII in cultured ECs, while Tie2 knockdown or blockade of Tie2 downstream PI3K/Akt pathway reduced COUP-TFII which could be reverted by the proteasome inhibition. Together, our results imply that Tie2 is essential for venous specification and maintenance via Akt mediated stabilization of COUP-TFII. DOI: http://dx.doi.org/10.7554/eLife.21032.001

[This corrects the article DOI: 10.1371/journal.pone.0064265.].

Introduction ADAMTS13 is a specific von Willebrand factor–cleaving protease. Severe deficiency of ADAMTS13 is the main cause of thrombotic thrombocytopenic purpura. ADAMTS13 is mainly synthesized and released from hepatic stellate cells and endothelial cells, but is also expressed in other cells, including kidney podocytes. Simvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, has a beneficial effect on atherosclerosis and also has anti-inflammatory and antithrombotic properties. A recent study indicates that ADAMTS13 reduces inflammatory plaque formation during early atherosclerosis in mice. In our study, we investigated the effects of simvastatin on inflammatory cytokines–induced ADAMTS13 expression in podocytes. Materials and Methods A conditionally immortalized mouse podocyte cell line was utilized to study the expression of ADAMTS13 in podocytes. The influence of TNF-α, IL-4, IL-6 and simvastatin on ADAMTS13 was investigated. ADAMTS13 mRNA levels in podocytes were measured by using real-time PCR and protein levels were detected by Western blotting. Results Simvastatin significantly up-regulated the expression levels of ADAMTS13 mRNA and protein in podocytes. IL-6 decreased ADAMTS13 expression, and TNF-α had no significant effects on ADAMTS13 expression in podocytes. IL-4 reduced ADAMTS13 mRNA expression but not its protein level. Simvastatin was able also reversed the inhibitory effect of IL-6. Conclusions We demonstrate that simvastatin increases the expression of ADAMTS13 in a dose-dependent manner in podocytes, which likely contributes to the antithrombotic property of statin. Different inflammatory cytokines have different effects on the levels of ADAMTS13 mRNA expression and protein within podocytes.