Subcellular distribution of α2-adrenoceptor subtypes in the rodent kidney.

Renal α2-adrenoceptors have been reported to play a role in the regulation of urinary output, renin secretion, and water and sodium excretion in the kidneys. However, the distribution of α2-adrenoceptor subtypes in the kidneys remains unclear. In this study, we aimed to investigate the localization of α2-adrenoceptor subtypes in rat kidneys using 8-week-old Sprague-Dawley rats. Immunofluorescence imaging revealed that both α2A- and α2B-adrenoceptors were expressed in the basolateral, but not apical, membrane of the epithelial cells of the proximal tubules. We also found that α2A- and α2B-adrenoceptors were not expressed in the glomeruli, collecting ducts, or the descending limb of the loop of Henle and vasa recta. In contrast, α2C-adrenoceptors were found to be localized in the glomeruli and lumen of the cortical and medullary collecting ducts. These results suggest that noradrenaline may act on the basement membrane of the proximal tubules through α2A- and α2B-adrenoceptors. Moreover, noradrenaline may be involved in the regulation of glomerular filtration and proteinuria through the induction of morphological changes in mesangial cells and podocytes via α2C-adrenoceptors. In the collecting ducts, urinary noradrenaline may regulate morphological changes of the microvilli through α2C-adrenoceptors. Our findings provide an immunohistochemical basis for understanding the cellular targets of α2-adrenergic regulation in the kidneys. This may be used to devise therapeutic strategies targeting α2-adrenoceptors.

Inhibition of α2-adrenoceptor is renoprotective in 5/6 nephrectomy-induced chronic kidney injury rats.

In the present study, we investigated the renoprotective effects of long-term treatment with yohimbine, an α2-adrenoceptor inhibitor, in a 5/6 nephrectomy-induced chronic kidney disease (CKD) rat model. Male Sprague-Dawley rats were randomly allocated into the following groups: sham-operated, 5/6-nephrectomized (5/6 Nx), 5/6 Nx + low or high dose of yohimbine (0.3 or 3.0 mg/L in drinking water, respectively), and 5/6 Nx + hydralazine (250 mg/L in drinking water). The 5/6 Nx group presented with renal dysfunction, hypertension, noradrenaline overproduction, and histopathological injuries. Blood pressure decreased in both the yohimbine- and hydralazine-treated groups. Treatment with high dose of yohimbine, but not hydralazine, apparently attenuated urinary protein excretion and noradrenaline concentration of renal venous plasma. Renal fibrosis and upregulated fibrosis-related gene expression were suppressed by high dose of yohimbine. Furthermore, yohimbine, but not hydralazine, treatment ameliorated the urinary concentration ability. These findings suggest that long-term yohimbine treatment can be a useful therapeutic option to prevent the progression of CKD.

Post-treatment with JP-1302 protects against renal ischemia/reperfusion-induced acute kidney injury in rats.

Ischemia/reperfusion injury is the most common cause of acute kidney injury. We previously revealed that pre-treatment with yohimbine or JP-1302 attenuated renal ischemia/reperfusion injury by inhibition of α2C-adrenoceptor antagonist. The aim of the present study is to investigate the effects of post-treatment with JP-1302 on renal ischemia/reperfusion injury in rats. Male Sprague Dawley rats were randomly divided into four groups: sham operation, ischemia/reperfusion, pre-treatment with JP-1302 (3.0 mg/kg) and post-treatment with JP-1302 groups. In ischemia/reperfusion injury, renal functional parameters, such as blood urea nitrogen, plasma creatinine and creatinine clearance, deteriorated after reperfusion. Renal venous norepinephrine concentrations, as well as inflammatory molecules in the kidney increased after reperfusion. Both pre- and post-treatment with JP-1302 improved renal dysfunction, tissue damage, renal venous norepinephrine concentrations and inflammatory molecules expression in the kidney. In conclusion, these results suggest that post-treatment with JP-1302 protects on ischemia/reperfusion-induced acute kidney injury by suppressing cytokine upregulation via α2C-adrenoceptors.

Specific protein-labeling and ligand-binding position analysis with amidopyrene probes as LDI MS tags.

To readily analyze the binding mode of protein-ligand interactions, we developed ligand-bound-type and ligand-dissociation-type probes having 6-amidopyrene (apy) as a detecting group. Matrix- and label-assisted laser desorption/ionization mass spectrometry (MALDI and LA-LDI MS) analyses and a covalent docking simulation using these probes precisely determined the binding position of the ligand biotin on the target protein avidin (RMSD = 0.786 and 0.332 Å). Our apy-probe-labeling method may be useful for determining the unknown ligand-binding sites of various target proteins.

Binding position analysis of target proteins with the use of amidopyrene probes as LA-LDI enhancing tags.

Amidopyrene-conjugated compounds can be detected by label-assisted laser desorption/ionization mass spectrometry (LA-LDI MS) without matrixes. When actin, a cytoskeletal protein, was labeled with an excess amount of amidopyrene N-hydroxysuccinate (apy-OSu), eight apy-labeled actin peptides were predominantly detected by LA-LDI MS. Then actin was labeled with an amidopyrene NHS ester of the antitumor marine macrolide aplyronine A (ApA-apy-OSu) to form a 1 : 1 conjugate. The sequence of an apy-labeled peptide was established as A(108)PLNPKANR(116) by MS/MS analysis, in which the NHS ester moiety specifically reacted with the ε-amino group of K113. While the fragmentation at the linker part reduces the detection sensitivity of apy-labeled peptides on LA-LDI MS, our chemical probe method is useful for analyzing the binding modes of various ligands and target biomacromolecules that include multiple and weak interactions.

Dexamethasone induces caveolin-1 in vascular endothelial cells: implications for attenuated responses to VEGF.

Steroids exert direct actions on cardiovascular cells, although underlying molecular mechanisms remain incompletely understood. We examined if steroids modulate abundance of caveolin-1, a regulatory protein of cell-surface receptor pathways that regulates the magnitudes of endothelial response to vascular endothelial growth factor (VEGF). Dexamethasone, a synthetic glucocorticoid, induces caveolin-1 at both levels of protein and mRNA in a time- and dose-dependent manner in pharmacologically relevant concentrations in cultured bovine aortic endothelial cells. Aldosterone, a mineralocorticoid, but not the sex steroids 17ß-estradiol, testosterone, or progesterone, elicits similar caveolin-1 induction. Caveolin-1 induction by dexamethasone and that by aldosterone were abrogated by RU-486, an inhibitor of glucocorticoid receptor, and by spironolactone, a mineralocorticoid receptor inhibitor, respectively. Dexamethasone attenuates VEGF-induced responses at the levels of protein kinases Akt and ERK1/2, small-G protein Rac1, nitric oxide production, and migration. When induction of caveolin-1 by dexamethasone is attenuated either by genetically by transient transfection with small interfering RNA or pharmacologically by RU-486, kinase responses to VEGF are rescued. Dexamethasone also increases expression of caveolin-1 protein in cultured human umbilical vein endothelial cells, associated with attenuated tube formation responses of these cells when cocultured with normal fibroblasts. Immunohistochemical analyses revealed that intraperitoneal injection of dexamethasone induces endothelial caveolin-1 protein in thoracic aorta and in lung artery in healthy male rats. Thus steroids functionally attenuate endothelial responses to VEGF via caveolin-1 induction at the levels of signal transduction, migration, and tube formation, identifying a novel point of cross talk between nuclear and cell-surface receptor signaling pathways.

Inhibition of microtubule assembly by a complex of actin and antitumor macrolide aplyronine A.

Aplyronine A (ApA) is a marine natural product that shows potent antitumor activity. While both ApA and ApC, a derivative of ApA that lacks a trimethylserine ester moiety, inhibit actin polymerization in vitro to the same extent, only ApA shows potent cytotoxicity. Therefore, the molecular targets and mechanisms of action of ApA in cells have remained unclear. We report that ApA inhibits tubulin polymerization in a hitherto unprecedented way. ApA forms a 1:1:1 heterotrimeric complex with actin and tubulin, in association with actin synergistically binding to tubulin, and inhibits tubulin polymerization. Tubulin-targeting agents have been widely used in cancer chemotherapy, but there are no previous descriptions of microtubule inhibitors that also bind to actin and affect microtubule assembly. ApA inhibits spindle formation and mitosis in HeLa S3 cells at 100 pM, a much lower concentration than is needed for the disassembly of the actin cytoskeleton. The results of the present study indicate that ApA represents a rare type of natural product, which binds to two different cytoplasmic proteins to exert highly potent biological activities.

Reduced expression of epidermal growth factor receptor, E-cadherin, and occludin in the skin of flaky tail mice is due to filaggrin and loricrin deficiencies.

Disruption of skin barrier function leads to increases in the percutaneous transfer of allergens and the incidence of atopic dermatitis. Flaky tail (Flg(ft)) mice have been used as a model of atopic dermatitis with skin barrier dysfunction. Although Flg(ft) mice are known to have filaggrin mutation, the mechanism responsible for the skin barrier dysfunction that they display needs to be determined, especially for the roles of epidermal adhesion and junction proteins. Herein, we report the decreased expression of epidermal growth factor receptor (EGFR), E-cadherin, occludin, and SIRT1 in the skin of Flg(ft) mice, compared with those in C57BL/6J mice. Administration of N-acetyl-L-cysteine, an antioxidant, in the drinking water improved these protein expressions in the skin of Flg(ft) mice. Notably, we discovered that loricrin expression was suppressed in Flg(ft) mice. In vitro experiments showed that filaggrin small interfering RNA, loricrin small interfering RNA, or SIRT1 inhibitor sirtinol suppressed the expression levels of EGFR, E-cadherin, and occludin in a human immortalized keratinocyte cell line (HaCaT cells). Our findings suggest that the observed reductions in EGFR, E-cadherin, and occludin expression were due to filaggrin deficiency accompanied with subsequent loricrin deficiency and disruption of the SIRT1 pathway in the skin of Flg(ft) mice.

Deranged epidermal differentiation in kl/kl mouse and the effects of ßKlotho siRNA on the differentiation of HaCaT cells.

Mice deficient in the klotho gene (kl/kl mice) display the phenotypes of human ageing. We found that the expression of epidermal differentiation-associated factors (keratin 1, keratin 10, filaggrin and loricrin) was lower in the skin of kl/kl mice than that of wild-type mice. In vitro experiments showed that the expression of ßKlotho, a family of klotho gene-encoded protein, was induced concomitantly with the differentiation of an immortalized human epidermal keratinocyte cell line (HaCaT cells) when they were cultured in an air-liquid interface. ßKlotho knockdown by small interfering ribonucleic acid suppressed the expression of the above differentiation-associated factors in HaCaT cells. ßKlotho small interfering ribonucleic acid increased the expression of keratin 14, which is expressed in mitotically active basal layer cells, and activated p44/p42 mitogen-activated protein kinase in the HaCaT cells grown in the air-liquid interface. These findings suggest that the epidermal differentiation is deranged in kl/kl mice, and ßKlotho is required for the differentiation of human epidermal keratinocytes.

Apoptosis-inducing activity of the actin-depolymerizing agent aplyronine A and its side-chain derivatives.

Aplyronine A (1) and mycalolide B (2), which are cytotoxic actin-depolymerizing marine macrolides, were revealed to induce apoptosis in human leukemia HL60 cells and human epithelial carcinoma HeLa S(3) cells. Based on these results, actin-depolymerizing compounds were expected to exhibit apoptosis-inducing activity in cancer cells. Compounds 3-6, which were synthesized based on the side-chain structure of aplyronine A, were evaluated for their actin-depolymerizing activities in vitro and cytotoxicities against HL60 cells. The growth-inhibitory activities of 3-6 were well correlated with their actin-depolymerizing activities, and derivative 6 was shown to induce the disruption of actin filaments and apoptosis in HL60 cells. These results suggested that actin-depolymerizing agents 1, 2, and 6-induced apoptosis in HL60 cells may have been due to their actin-depolymerizing activity.

Interactions of the antitumor macrolide aplyronine A with actin and actin-related proteins established by its versatile photoaffinity derivatives.

The antitumor and apoptogenic macrolide aplyronine A (ApA) is a potent actin-depolymerizing agent. We developed an ApA acetylene analog that bears the aryldiazirine group at the C34 terminus, which formed a covalent bond with actin. With the use of the photoaffinity biotin derivatives of aplyronines A and C, Arp2 and Arp3 (actin-related proteins) were specifically purified as binding proteins along with actin from tumor cell lysate. However, Arp2 and Arp3 did not covalently bind to aplyronine photoaffinity derivatives. Thus, actin-related proteins might indirectly bind to ApA as the ternary adducts of the actin/ApA complex or through the oligomeric actin.

A case of Degos disease successfully treated with corticosteroid combined with cyclophosphamide.

Degos disease is a rare disorder characterized by systemic vasculitis involving various organs. There is no established, effective treatment for the disorder, and its prognosis is still poor. Combination therapy with corticosteroid and cyclophosphamide is considered effective for vasculitides involving the small arteries such as ANCA-associated vasculitis. We present here a 42-year-old man who developed Degos disease over several months, and was successfully treated using combined treatment with corticosteroid and cyclophosphamide.

Novel Antibody Exerts Antitumor Effect through Downregulation of CD147 and Activation of Multiple Stress Signals.

CD147 is an immunoglobulin-like receptor that is highly expressed in various cancers and involved in the growth, metastasis, and activation of inflammatory pathways via interactions with various functional molecules, such as integrins, CD44, and monocarboxylate transporters. Through screening of CD147-targeting antibodies with antitumor efficacy, we discovered a novel rat monoclonal antibody #147D. This humanized IgG4-formatted antibody, h4#147D, showed potent antitumor efficacy in xenograft mouse models harboring the human PDAC cell line MIA PaCa-2, HCC cell line Hep G2, and CML cell line KU812, which featured low sensitivity to the corresponding standard-of-care drugs (gemcitabine, sorafenib, and imatinib, respectively). An analysis of tumor cells derived from MIA PaCa-2 xenograft mice treated with h4#147D revealed that cell surface expression of CD147 and its binding partners, including CD44 and integrin α3ß1/α6ß1, was significantly reduced by h4#147D. Inhibition of focal adhesion kinase (FAK), activation of multiple stress responsible signal proteins such as c-JunN-terminal kinase (JNK) and mitogen-activated protein kinase p38 (p38MAPK), and expression of SMAD4, as well as activation of caspase-3 were obviously observed in the tumor cells, suggesting that h4#147D induced tumor shrinkage by inducing multiple stress responsible signals. These results suggest that the anti-CD147 antibody h4#147D offers promise as a new antibody drug candidate.

Activation of vascular endothelial growth factor receptor 2 in a cellular model of loricrin keratoderma.

Loricrin is a major constituent of the epidermal cornified cell envelope. Recently, heterozygous loricrin gene mutations have been identified in two dominantly inherited skin diseases, Vohwinkel syndrome with ichthyosis and progressive symmetric erythrokeratoderma, collectively termed loricrin keratoderma. We generated stable HaCaT cell lines that express wild-type (WT) loricrin and a mutant form found in Vohwinkel syndrome with ichthyosis, using an ecdysone-inducible promoter system. The cells expressing the mutant loricrin grew more rapidly than those expressing WT loricrin after induction for 5 days. Confocal immunofluorescence microscopy revealed that phospho-Akt occurred in the nucleolus where the mutant loricrin was also located. The level of activity of Akt kinase was about nine times higher in cells with the mutant than in those with WT loricrin. ERK1/2, the epidermal growth factor receptor, vascular endothelial growth factor (VEGF) receptor 2 and Stat3 were all phosphorylated in cells with the mutant loricrin. The docking proteins, Gab1 and c-Cbl, were also tyrosine-phosphorylated in these cells. Furthermore, chromatin immunoprecipitation assays showed that Stat3 protein bound to the VEGF promoter in cells with the mutant. Thus, this study suggests that VEGF release and the subsequent activation of VEGF receptor 2 link loricrin gene mutations to rapid cell proliferation in a cellular model of loricrin keratoderma.

Lipid peroxidation-induced VEGF expression in the skin of KKAy obese mice.

Obesity is known to be associated with a number of effects on skin physiology. KKA(y) obese mouse is a model of type 2 diabetes characterized by systemic oxidative stress because of severe obesity, hypertriglyceridaemia, hyperglycaemia and hyperinsulinaemia. We investigated lipid peroxidation and vascular endothelial growth factor (VEGF) expression in the skin of KKA(y) obese mice. We also investigated the effect of lipid peroxidation derivatives on VEGF production and proliferation in human epidermal keratinocyte cell line (HaCaT). The lipid peroxidation level in the mouse skin tissue was determined by measuring the levels of thiobarbituric acid-reactive substances. The levels of VEGF expression, p44/p42 mitogen-activated protein kinase (MAPK) activation and CD36 expression were analysed by Western blot. Their localization was examined by immunofluorescence. For the in vitro experiments, an enzyme-linked immunosorbent assay was utilized to measure VEGF secretion in the medium. In vitro experiments demonstrated that lipid peroxidation derivatives increased VEGF production in HaCaT cells, which was blocked by a p44/p42 MAPK inhibitor and anti-CD36 antibody. We observed increased levels of lipid peroxidation derivatives, p44/p42 MAPK activation and VEGF expression in the skin of KKA(y) obese mice. Notably, pitavastatin, an inhibitor of competitive 3-hydroxy-3-methylglutaryl coenzyme A reductase, suppressed all of these processes. Our results suggest that lipid peroxidation induces VEGF expression via CD36 and p44/p42 MAPK pathway in the skin of obese mice.

A founder effect of c.1938delC in ITGB4 underlies junctional epidermolysis bullosa and its application for prenatal testing.

Junctional epidermolysis bullosa associated with pyloric atresia (JEB-PA) is one of the most severe inherited skin diseases, characterized by generalized blister formation and occlusion of the pylorus at birth. Most JEB-PA patients have mutations in the gene encoding ß4 integrin (ITGB4). No recurrent mutations in ITGB4 have been described as having founder effects. We collected three JEB-PA families with c.1938delC in ITGB4. Haplotype analysis using single nucleotide polymorphism markers throughout ITGB4 suggested one rare haplotype (2.8% of the Han Chinese and ethnic Japanese populations) in all alleles with c.1938delC. The parents of one of the three families sought prenatal diagnosis for a subsequent pregnancy. We succeeded in performing prenatal exclusion of JEB-PA using the foetal genomic DNA. Our study clearly demonstrated that recurrent c.1938delC in ITGB4 is a founder mutation in JEB-PA patients, and that genotyping of the mutation can be utilized for prenatal diagnosis of JEB-PA.

Japanese guidelines for the management of palmoplantar keratoderma.

Palmoplantar keratoderma (PPK) is a collective term for keratinizing disorders in which the main clinical symptom is hyperkeratosis on the palms and soles. To establish the first Japanese guidelines approved by the Japanese Dermatological Association for the management of PPKs, the Committee for the Management of PPKs was founded as part of the Study Group for Rare Intractable Diseases. These guidelines aim to provide current information for the management of PPKs in Japan. Based on evidence, they summarize the clinical manifestations, pathophysiologies, diagnostic criteria, disease severity determination criteria, treatment, and treatment recommendations. Because of the rarity of PPKs, there are only few clinical studies with a high degree of evidence. Therefore, several parts of these guidelines were established based on the opinions of the committee. To further optimize the guidelines, periodic revision in line with new evidence is necessary.

Symmetrical Skin Lesions on the Gluteal Region in a Patient with Anti-Laminin-332 Mucous Membrane Pemphigoid.

Mucous membrane pemphigoid (MMP), previously called cicatricial pemphigoid, is a rare subepidermal immunobullous disorder that primarily affects the mucous membranes (1,2). MMP is divided into two major subtypes, anti-BP180-type MMP and anti-laminin-332 (previously called laminin 5 or epiligrin) MMP. Anti-laminin-332 MMP is known to be associated with malignant tumors (3), which may cause overexpression of autoantibodies and induce autoimmunity to laminin-332 (4). MMP primarily affects the mucous membranes, and widespread skin lesions are rare. In MMP, circumscribed skin lesions have been previously reported as occurring on the head, neck, and upper trunk (5). We report a case of anti-laminin-332 MMP presenting with symmetrical skin lesions characteristic of MMP on the weight-bearing areas of the gluteal region. A 66-year-old Japanese man presented with a month-long history of multiple erosions and blisters on the mucous membranes and skin, with conjunctival hyperemia, nasal obstruction, oral pain, and hoarseness of voice. Three days before the first visit, he was diagnosed with gastric cancer with liver metastasis by gastrointestinal endoscopy and abdominal ultrasound examination for tarry stool. Physical examination demonstrated erosions and tense bullae on the conjunctivae, tongue, and lips (Figure 1, a,b), as well as erosive erythematous skin lesions on the nape, right index finger, both legs, and symmetric lesions on the gluteal region (Figure 1, c). His body weight was 86 kg. Laboratory examinations showed slight liver dysfunction and elevation of C-reactive protein levels. Histopathologic examination of the skin lesions demonstrated subepidermal blisters with lymphocytic and eosinophilic infiltrates (Figure 1, d,e). Direct immunofluorescence (IF) revealed linear deposits of IgG and C3, but not IgA, along the basement membrane zone (BMZ) (Figure 1, f,g). An IgG subclass study showed IgG1 and IgG4 deposits. Indirect IF on normal human skin revealed weak positivity for IgA anti-keratinocyte cell surface antibodies and IgG anti-BMZ antibodies, which were bound to the dermal side of 1 mol/L NaCl-split skin (Figure 1, h). IgG immunoblot analyses of both normal human epidermal and dermal extracts showed negative results (including BP230, BP180, 290 kDa type VII collagen, and 200 kDa laminin-γ1). Immunoprecipitation using radio-labeled cultured keratinocyte lysate demonstrated positive reactivity with laminin-332 (Figure 1, i). We established the diagnosis of anti-laminin-332 MMP. We started treatment with oral minocycline (200 mg/day) and niacinamide (900 mg/day) with topical corticosteroids without any effect after 2 weeks of therapy. Administration of oral prednisolone (40 mg/day) with topical corticosteroids and alprostadil ointment on the skin lesions, as well as beclometasone dipropionate powder on the oral lesions resulted in significant improvement of mucocutaneous lesions within 10 days. Although the gastric cancer and liver metastasis initially responded to chemotherapy with fluorouracil and cisplatin, the patient succumbed to multiple organ failure 9 months after the initial visit. Anti-laminin-332 antibodies were originally detected by immunoprecipitation, as in our case. Immunoblotting of purified human laminin-332 have been subsequently developed, which detects the 165/145 kDa α3, 140 kDa ß3, and 105 kDa γ2 subunits of laminin-332 in various patterns (6). Today, the ELISA system uses laminin-332 preparations as adjunct diagnostic tools in MMP (7). Occasionally, a wide spectrum of autoantibodies is detected in MMP, for example, MMP with IgG antibodies to both BP180 and laminin-332, which were considered to be developed via epitope spreading. Detection of circulating IgA autoantibodies against the skin have also been reported in MMP (8). However, the pathogenic significance and mechanisms of coexistence of IgG anti-laminin-332 antibodies and IgA anti-keratinocyte cell surface antibodies found in our case are currently unknown. It is generally considered that IgG1 antibodies activate complements and are pathogenic in MMP, while IgG4 antibodies behave as blocking antibodies and are protective. In our case, direct IF revealed IgG1 and IgG4 deposits; the same was reported in a previous case report (9). The pathogenic roles of autoantibodies with different IgG subclasses need to be analyzed in further studies. Conjunctival mucosal lesions in MMP may occur by rubbing of the eyes due to irritation. Blinking subjects the conjunctivae to repeated friction. Vocal cords vibrate during breathing and speaking. The tongue moves while eating and drinking; in particular, the tip of the tongue gets into frequent contact with the inner sides of the incisor teeth. In the present case, characteristic symmetrical skin lesions were seen on the weight-bearing areas of the gluteal region on bony prominences which receive mechanical stresses in the sitting position. These skin lesions were subjected to repeated stretch and pressure stresses, but no ischemic changes were observed, such as decubitus ulcers. Therefore, the symmetrical skin lesions in the gluteal region as well as the ocular and oral mucosal lesions seen in our patient might have resulted from the same mechanism of pathogenesis. We reported a case of anti-laminin-332 MMP presenting with symmetrical gluteal skin lesions, probably induced by mechanical stress. MMP primarily affects the mucous membranes, and widespread skin lesions are rare. Our case emphasizes that clinicians need to specifically check for the presence of skin lesions on weight-bearing parts of the body during examination of patients with suspected MMP.