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Serine proteases are proteolytic enzymes that are involved in the regulation of various physiological processes. We generated mice lacking the membrane-anchored channel-activating serine protease (CAP) 1 (also termed protease serine S1 family member 8 [Prss8] and prostasin) in skin, and these mice died within 60 h after birth. They presented a lower body weight and exhibited severe malformation of the stratum corneum (SC). This aberrant skin development was accompanied by an impaired skin barrier function, as evidenced by dehydration and skin permeability assay and transepidermal water loss measurements leading to rapid, fatal dehydration. Analysis of differentiation markers revealed no major alterations in CAP1/Prss8-deficient skin even though the epidermal deficiency of CAP1/Prss8 expression disturbs SC lipid composition, corneocyte morphogenesis, and the processing of profilaggrin. The examination of tight junction proteins revealed an absence of occludin, which did not prevent the diffusion of subcutaneously injected tracer (∼600 D) toward the skin surface. This study shows that CAP1/Prss8 expression in the epidermis is crucial for the epidermal permeability barrier and is, thereby, indispensable for postnatal survival.

Elevated plasma urate levels are associated with metabolic, cardiovascular, and renal diseases. Urate may also form crystals, which can be deposited in joints causing gout and in kidney tubules inducing nephrolithiasis. In mice, plasma urate levels are controlled by hepatic breakdown, as well as, by incompletely understood renal processes of reabsorption and secretion. Here, we investigated the role of the recently identified urate transporter, Glut9, in the physiological control of urate homeostasis using mice with systemic or liver-specific inactivation of the Glut9 gene. We show that Glut9 is expressed in the basolateral membrane of hepatocytes and in both apical and basolateral membranes of the distal nephron. Mice with systemic knockout of Glut9 display moderate hyperuricemia, massive hyperuricosuria, and an early-onset nephropathy, characterized by obstructive lithiasis, tubulointerstitial inflammation, and progressive inflammatory fibrosis of the cortex, as well as, mild renal insufficiency. In contrast, liver-specific inactivation of the Glut9 gene in adult mice leads to severe hyperuricemia and hyperuricosuria, in the absence of urate nephropathy or any structural abnormality of the kidney. Together, our data show that Glut9 plays a major role in urate homeostasis by its dual role in urate handling in the kidney and uptake in the liver.gout ͉ knockout ͉ nephrolithiasis ͉ uric acid

T he cardiac action potential (AP) is initiated by the Na + channel Na V 1.5, an established key element for cardiac excitability and impulse propagation. The importance of Na V 1.5 is exemplified by the myriad of cardiac disorders caused by hundreds of mutations identified in SCN5A, the gene coding for Na V 1.5.1 For some SCN5A mutation carriers, cardiac conduction slowing or block, secondary to reduced Na + channel function, predisposes them to ventricular arrhythmias and sudden cardiac death. Editorial see p 132 Clinical Perspective on p 160The cardiac Na + channel is composed of a 220-kDa α-subunit, Na V 1.5, constituting the pore of the channel, which is known to associate with four ≈30-kDa β-subunits. Recent studies have demonstrated that many proteins interact with and regulate Na V 1.5. 2 The physiological relevance of these interactions, however, is poorly understood, mainly due to a lack of in vivo studies. Many protein-protein interaction motifs for these regulatory proteins are located at the C-terminus of Na V 1.5. 2 In particular, we have previously demonstrated that Na V 1.5 associates with the dystrophinsyntrophin multiprotein complex (DMC) in cardiac cells.3 InBackground-Sodium channel Na V 1.5 underlies cardiac excitability and conduction. The last 3 residues of Na V 1.5 (Ser-IleVal) constitute a PDZ domain-binding motif that interacts with PDZ proteins such as syntrophins and SAP97 at different locations within the cardiomyocyte, thus defining distinct pools of Na V 1.5 multiprotein complexes. Here, we explored the in vivo and clinical impact of this motif through characterization of mutant mice and genetic screening of patients. Methods and Results-To investigate in vivo the regulatory role of this motif, we generated knock-in mice lacking the SIV domain (∆SIV). ∆SIV mice displayed reduced Na V 1.5 expression and sodium current (I Na ), specifically at the lateral myocyte membrane, whereas Na V 1.5 expression and I Na at the intercalated disks were unaffected. Optical mapping of ∆SIV hearts revealed that ventricular conduction velocity was preferentially decreased in the transversal direction to myocardial fiber orientation, leading to increased anisotropy of ventricular conduction. Internalization of wild-type and ΔSIV channels was unchanged in HEK293 cells. However, the proteasome inhibitor MG132 rescued ΔSIV I Na , suggesting that the SIV motif is important for regulation of Na V 1.5 degradation. A missense mutation within the SIV motif (p.V2016M) was identified in a patient with Brugada syndrome. The mutation decreased Na V 1.5 cell surface expression and I Na when expressed in HEK293 cells. Conclusions-Our results demonstrate the in vivo significance of the PDZ domain-binding motif in the correct expression of Na V 1.5 at the lateral cardiomyocyte membrane and underline the functional role of lateral Na V 1.5 in ventricular conduction. Recherche 1087, L'Institut du Thorax, Nantes, France (R.R.); Centre National de la Recherche Scientifique Unité Mixte de Recherche 6291, Nantes, France...

Altered serine protease activity is associated with skin disorders in humans and in mice. The serine protease channel-activating protease-1 (CAP1; also termed protease serine S1 family member 8 (Prss8)) is important for epidermal homeostasis and is thus indispensable for postnatal survival in mice, but its roles and effectors in skin pathology are poorly defined. In this paper, we report that transgenic expression in mouse skin of either CAP1/Prss8 (K14-CAP1/Prss8) or protease-activated receptor-2 (PAR2; Grhl3PAR2/+), one candidate downstream target, causes epidermal hyperplasia, ichthyosis and itching. K14-CAP1/Prss8 ectopic expression impairs epidermal barrier function and causes skin inflammation characterized by an increase in thymic stromal lymphopoietin levels and immune cell infiltrations. Strikingly, both gross and functional K14-CAP1/Prss8-induced phenotypes are completely negated when superimposed on a PAR2-null background, establishing PAR2 as a pivotal mediator of pathogenesis. Our data provide genetic evidence for PAR2 as a downstream effector of CAP1/Prss8 in a signalling cascade that may provide novel therapeutic targets for ichthyoses, pruritus and inflammatory skin diseases.

The localization and significance of regulatory T cells (Treg) in allograft rejection is of considerable clinical and immunological interest. We analyzed 80 human renal transplant biopsies (including seven donor biopsies) with a double immunohistochemical marker for the Treg transcription factor FOXP3, combined with a second marker for CD4 or CD8. Quantitative FOXP3 cell counts were performed and analyzed for clinical and pathologic correlates. FOXP3+ cells were present in the interstitium in acute cellular rejection (ACR) type I and II, at a greater density than in acute humoral rejection or CNI toxicity (p < 0.01). Most FOXP3 + cells were CD4 + (96%); a minority expressed CD8. FOXP3 + CD4 + cells were concentrated in the tubules (p < 0.001), suggesting a selective attraction or generation at that site. Considering only patients with ACR, a higher density of FOXP3 + correlated with HLA class II match (p = 0.03), but paradoxically with worse graft survival. We conclude that infiltration of FOXP3 + cells occurs in ACR to a greater degree than in humoral rejection, however, within the ACR group, no beneficial effect on outcome was evident. Tregs concentrate in tubules, probably contributing to FOXP3 mRNA in urine; the significance and pathogenesis of 'Treg tubulitis' remains to be determined.

Genetic defects in autosomal-dominant polycystic kidney disease (ADPKD) promote cystic growth of renal tubules, at least in part by stimulating the accumulation of cAMP. How renal cAMP levels are regulated is incompletely understood. We show that cAMP and the expression of its synthetic enzyme adenylate cyclase-6 (AC6) are up-regulated in cystic kidneys of Bicc1(-)(/-) knockout mice. Bicc1, a protein comprising three K homology (KH) domains and a sterile alpha motif (SAM), is expressed in proximal tubules. The KH domains independently bind AC6 mRNA and recruit the miR-125a from Dicer, whereas the SAM domain enables silencing by Argonaute and TNRC6A/GW182. Bicc1 similarly induces silencing of the protein kinase inhibitor PKIα by miR-27a. Thus, Bicc1 is needed on these target mRNAs for silencing by specific miRNAs. The repression of AC6 by Bicc1 might explain why cysts in ADPKD patients preferentially arise from distal tubules.

The contribution of neuropeptide Y (NPY), deriving from adrenal medulla, to the adrenosympathetic tone is unknown. We found that in response to NPY, primary cultures of mouse adrenal chromaffin cells secreted catecholamine, and that this effect was abolished in cultures from NPY Y1 receptor knockout mice (Y1؊͞؊). Compared with wild-type mice (Y1؉͞؉), the adrenal content and constitutive release of catecholamine were increased in chromaffin cells from Y 1؊͞؊ mice. In resting animals, catecholamine plasma concentrations were higher in Y1؊͞؊ mice. Comparing the adrenal glands of both genotypes, no differences were observed in the area of the medulla, cortex, and X zone. The high turnover of adrenal catecholamine in Y 1؊͞؊ mice was explained by the enhancement of tyrosine hydroxylase (TH) activity, although no change in the affinity of the enzyme was observed. The molecular interaction between the Y 1 receptor and TH was demonstrated by the fact that NPY markedly inhibited the forskolin-induced luciferin activity in Y 1 receptor-expressing SK-N-MC cells transfected with a TH promoter sequence. We propose that NPY controls the release and synthesis of catecholamine from the adrenal medulla and consequently contributes to the sympathoadrenal tone. (3,4). NPY is an important neurotransmitter of the sympathetic function that potentiates the catecholamine vasoconstrictor activity through the Y 1 receptor and exerts prejunctional inhibitory effects on NE release from the sympathetic nerve endings of the heart through the Y 2 receptor (5). In addition, the nerve terminals of parasympathetic neurons in the mouse heart possess Y 2 receptors, which, when activated, reduce acetylcholine release, also causing an inhibition of the parasympathetic nervous system (6). We have shown that NPY Y 1 knockout mice (Y 1 Ϫ͞Ϫ) lose their ability to potentiate NE-induced vasoconstriction and have normal blood pressure, probably indicating a minor role of NPY in the maintenance of blood pressure homeostasis (7). Recently, these mice were investigated for their cardiac sympathovagal balance in baseline conditions and during an acute social challenge. Reduced somatomotor activity during nonsocial challenges, lower heart rate in baseline conditions, and larger heart rate responsiveness during social defeat were reported (8). Besides its presence in nerve endings, NPY is produced by chromaffin cells of adrenal medulla of different species, including human (9). The mouse has higher adrenal NPY content than rat, pig, or humans (10, 11). The effect of NPY on the adrenal medulla is controversial. NPY stimulated catecholamine release from intact rat adrenal capsular tissue (12), although an inhibitory effect of NPY on catecholamine secretion in rat adrenomedullary primary cell cultures was also observed (13). Moreover, there is a weak inhibitory effect of NPY on NE and epinephrine (EP) release from bovine chromaffin cells, evoked by addition of a cholinergic agonist (14, 15). However, depending on the experimental conditions, conflicting results wer...