RESUMO
Recent findings suggest that uncarboxylated osteocalcin (GluOC) promotes glucose and lipid metabolism via its putative receptor GPRC6A; however, its direct effect on adipocytes remains elusive. In this study, we elucidated the effects of GluOC on adipocytes, with an emphasis on the role of cell adhesion molecules. We determined that GluOC promoted the expression of adipocyte adhesion molecule (ACAM) and its transcription factor Krüppel-like factor 4 and enhanced the cortical actin filament assembly, which ameliorated lipid droplet hypertrophy. Additionally, GluOC upregulated the expression of integrin αVß3 and activation of focal adhesion kinase (FAK) and prevented insulin receptor substrate 1 (IRS1) degradation by inhibiting the ubiquitin-proteasome system via the FAK-PLC-PKC axis, which activated IRS1-Akt-mediated glucose transporter 4 (GLUT4) transport. Furthermore, we showed that GluOC elevated the expression of the insulin-independent glucose transporters GLUT1 and GLUT8, which facilitated insulin stimulation-independent glucose transport. The GluOC-induced activation of integrin αVß3 signaling promoted microtubule assembly, which improved glucose and lipid metabolism via its involvement in intracellular vesicular transport. GluOC treatment also suppressed collagen type 1 formation, which might prevent adipose tissue fibrosis in obese individuals. Overall, our results imply that GluOC promotes glucose and lipid metabolism via ACAM, integrin αVß3, and GLUT1 and 8 expression, directly affecting adipocytes.
Assuntos
Glucose , Metabolismo dos Lipídeos , Humanos , Glucose/metabolismo , Osteocalcina/metabolismo , Osteocalcina/farmacologia , Metabolismo dos Lipídeos/genética , Transportador de Glucose Tipo 1/metabolismo , Integrina alfaVbeta3 , Adipócitos/metabolismo , Insulina/metabolismo , Moléculas de Adesão Celular/metabolismoRESUMO
c-Jun NH2-terminal protein kinase (JNK) and p38 are stress-activated mitogen-activated protein kinases (MAPK) that are phosphorylated by various stimuli. It has been reported that the loss of desmoglein (DSG) 3, a desmosomal transmembrane core molecule, in keratinocytes impairs cell-cell adhesion accompanied by p38 MAPK activation. To understand the biological role of DSG3 in desmosomes and its relationship with stress-activated MAPKs, we established DSG3 knockout keratinocytes (KO cells). Wild-type cells showed a linear localization of DSG1 to cell-cell contacts, whereas KO cells showed a remarkable reduction despite the increased protein levels of DSG1. Cell-cell adhesion in KO cells was impaired over time, as demonstrated by dispase-based dissociation assays. The linear localization of DSG1 to cell-cell contacts and the strength of cell-cell adhesion were promoted by the pharmacological inhibition of JNK. Conversely, pharmacological activation of JNK, but not p38 MAPK, in wild-type cells reduced the linear localization of DSG1 in cell-cell contacts. Our data indicate that DSG1 and DSG2 in KO cells cannot compensate for the attenuation of cell-cell adhesion strength caused by DSG3 deficiency and that JNK inhibition restores the strength of cell-cell adhesion by increasing the linear localization of DSG1 in cell-cell contacts in KO cells. Inhibition of JNK signaling may improve cell-cell adhesion in diseases in which DSG3 expression is impaired.
Assuntos
Desmogleína 3 , Queratinócitos , Adesão Celular/genética , Desmogleína 3/genética , Desmogleína 3/metabolismo , Queratinócitos/metabolismo , Sistema de Sinalização das MAP QuinasesRESUMO
Pleuroparenchymal fibroelastosis is a recently recognized clinical entity characterized by interstitial pneumonia with proliferating elastin in the upper lung regions. Pleuroparenchymal fibroelastosis is categorized as idiopathic or reported depending on the coexistent initiating factors; however, congenital contractural arachnodactyly, which is caused by abnormal production of elastin based on a mutation in the fibrillin-2 gene, is rarely reported with lung lesion resembling pleuroparenchymal fibroelastosis. We present a case of pleuroparenchymal fibroelastosis in a patient with a novel mutation in the fibrillin-2 gene, which encodes the prenatal fibrillin-2 protein as a scaffold for elastin.
RESUMO
Retinoic acid (RA) plays an important role in epithelial homeostasis and influences the morphology, proliferation, differentiation and permeability of epithelial cells. Mouse keratinocytes, K38, reconstituted non-keratinized stratified epithelium in three-dimensional (3D) cultures with serum, which contains retinol (a source of RA), but the morphology was different from in vivo epithelium. The formed epithelium was thick, with loosened cell-cell contacts. Here, we investigated whether the inhibition of RA receptor (RAR)/retinoid X receptor (RXR)-mediated signaling by an RXR antagonist, HX 531, improved K38 3D cultures in terms of morphology and intercellular junctions. The epithelium formed by 0.5 µM HX531 was thin, and the intercellular space was narrowed because of the restoration of the layer-specific distribution of desmoglein (DSG)-1, DSG3 and plakoglobin (PG). Moreover, the levels of desmosomal proteins and tight junction proteins, including DSG1, DSG2, DSG3, PG, claudin (CLDN)-1 and CLDN4 increased, but the adherens junction protein, E-cadherin, did not show any change. Furthermore, CLDN1 was recruited to occludin-positive cell-cell contacts in the superficial cells and transepithelial electrical resistance was increased. Therefore, K38 3D cultures treated with 0.5 µM HX531 provides a useful in vitro model to study intercellular junctions in the non-keratinized epithelium.
Assuntos
Caderinas de Desmossomos , Queratinócitos , Receptores X de Retinoides , Animais , Benzoatos/farmacologia , Compostos de Bifenilo/farmacologia , Técnicas de Cultura de Células em Três Dimensões , Caderinas de Desmossomos/metabolismo , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Camundongos , Permeabilidade , Receptores X de Retinoides/antagonistas & inibidores , Receptores X de Retinoides/metabolismoRESUMO
In this study, we examined the effects of calyculin A, a phosphatase inhibitor, on motility, protein phosphorylation, and the distribution of phospho-(Ser/Thr) PKA substrates in frozen-thawed bull spermatozoa that are actually used by most farmers for breeding. The data showed that calyculin A, which has been reported to have a positive effect on the motility of ejaculated fresh spermatozoa, distinctly decreased the motility of frozen-thawed bull spermatozoa even if a cell activator, such as caffeine, was present in the incubation medium and that the suppressive effect of calyculin A was dose-dependent and continued for at least 200 min. Immunoblot analyses revealed that de novo protein phosphorylation was not detected in spermatozoa exposed to caffeine or dbcAMP (a cell-permeable cAMP analog), while the addition of calyculin A to the medium brought about the appearance of several phosphorylated proteins at 50 kDa and 75 kDa, suggesting that 50 kDa and 75 kDa proteins, which were phosphorylated by activation of cAMP-dependent PKA, were not dephosphorylated and were accumulated in spermatozoa due to the suppression of calyculin A-sensitive protein phosphatases. Immunofluorescence microscopy revealed that calyculin A caused, alone or in conjunction with caffeine or dbcAMP, the accumulation of phospho-PKA substrates at the annulus, although caffeine or dbcAMP alone did not. This study suggested that calyculin A decreases the motility of frozen-thawed bull spermatozoa concomitant with the accumulation of phospho-(Ser/Thr) PKA substrates at the annulus of flagella.
Assuntos
AMP Cíclico , Motilidade dos Espermatozoides , Animais , Bovinos , Criopreservação , AMP Cíclico/metabolismo , Masculino , Toxinas Marinhas , Oxazóis , Fosforilação , EspermatozoidesRESUMO
Bone provides skeletal support and functions as an endocrine organ by producing osteocalcin, whose uncarboxylated form (GluOC) increases the metabolism of glucose and lipid by activating its putative G protein-coupled receptor (family C group 6 subtype A). Low doses (≤10 ng/ml) of GluOC induce the expression of adiponectin, adipose triglyceride lipase and peroxisome proliferator-activated receptor γ, and promote active phosphorylation of lipolytic enzymes such as perilipin and hormone-sensitive lipase via the cAMP-PKA-Src-Rap1-ERK-CREB signaling axis in 3T3-L1 adipocytes. Administration of high-dose (≥20 ng/ml) GluOC induces programmed necrosis (necroptosis) through a juxtacrine mechanism triggered by the binding of Fas ligand, whose expression is induced by forkhead box O1, to Fas that is expressed in adjacent adipocytes. Furthermore, expression of adiponectin and adipose triglyceride lipase in adipocytes is triggered in the same manner as following low-dose GluOC stimulation; these effects protect mice from diet-induced accumulation of triglycerides in hepatocytes and consequent liver injury through the upregulation of nuclear translocation of nuclear factor-E2-related factor-2, expression of antioxidant enzymes, and inhibition of the c-Jun N-terminal kinase pathway. Evaluation of these molecular mechanisms leads us to consider that GluOC might have potential as a treatment for lipid metabolism disorders. Indeed, there have been many reports demonstrating the negative correlation between serum osteocalcin levels and obesity or non-alcoholic fatty liver disease, a common risk factor for which is dyslipidemia in humans. The present review summarizes the effects of GluOC on lipid metabolism as well as its possible therapeutic application for metabolic diseases including obesity and dyslipidemia.
Assuntos
Tecido Adiposo/metabolismo , Metabolismo dos Lipídeos/fisiologia , Fígado/metabolismo , Osteocalcina/fisiologia , Adiponectina/metabolismo , Tecido Adiposo/citologia , Animais , Humanos , Camundongos , Necroptose , Osteocalcina/metabolismo , Transdução de SinaisRESUMO
Although emergence of keratin 17 (K17) and reciprocal loss of K13 are immunohistochemical hallmarks for oral mucosal malignancy, we report here findings of K17-positive (+) speckles, possibly equivalent to Civatte bodies, in benign oral lichen planus. Sixty-two biopsy samples from oral lichen planus cases were subjected to immunohistochemical examinations to analyze the distribution as well as histopathogenesis of Civatte bodies. K17 was irregularly positive among oral lichen planus-affected epithelial cells, and K17-positive (+) filamentous structures were irregularly distributed within the cytoplasm in confocal images. K17+ speckles were identified as Civatte bodies, and they were mainly distributed in the interface between epithelial cells and lymphocytic infiltrates (type A, 52.8%), followed by distribution within the epithelial layer (type B, 24.7%) or within the lamina propria with lymphocytic infiltration (type C, 22.5%). Apoptotic figures were often engulfed by macrophages and clearly distinguished from Civatte bodies by the presence TUNEL signals. These results indicate that K17 is a sensitive immunohistochemical marker for Civatte bodies and useful for differential diagnosis of oral lichen planus from other oral mucosal lesions. Civatte bodies are generated from denucleation of K17+ epithelial cells during the process of cell death via dyskeratosis, which is possibly related to blood capillary collapse.
Assuntos
Coloides/análise , Queratina-17/metabolismo , Líquen Plano Bucal/patologia , Mucosa Bucal/patologia , Pele/patologia , Estudos de Casos e Controles , Humanos , Líquen Plano Bucal/metabolismo , Mucosa Bucal/metabolismo , Pele/metabolismoRESUMO
We isolated the transmembrane and coiled-coil domains 2 (Tmco2) gene using a polymerase chain reaction-based subtraction technique. Tmco2 is predominantly expressed in rat testes starting from 4 weeks of age. Rat TMCO2 consists of 187 amino acids with a predicted molecular mass of 20.6 kDa. When expressed in COS7 cells, TMCO2 was found as vesicle-like structures in the cytoplasm, whereas TMCO2ΔTM lacking the transmembrane (TM) region was found diffused in the cytoplasm. These results suggest that the TM region in TMCO2 is essential for its specificity of localization. Immunocytochemical analyzes indicated that rat TMCO2 was localized as small semiluminate bodies or cap-like structures in the vicinity of round spermatid nuclei and as curved lines associated with nuclei of elongated spermatids and caput epididymal spermatozoa. However, it was detected in only a small part of cauda epididymal spermatozoa. Double immunolabeling of the spermatids and spermatozoa with the anti-TMCO2 antibody and the monoclonal anti-MN7 antibody showed that TMCO2 was predominantly associated with the inner acrosomal membrane in spermatids and caput epididymal spermatozoa. Our findings suggest that TMCO2 might be involved in the process of acrosome biogenesis, especially binding of acrosome to a nucleus, during spermiogenesis.
RESUMO
Keratinocytes take up serum-derived retinol (vitamin A) and metabolize it to all-trans-retinoic acid (atRA), which binds to the nuclear retinoic acid receptor (RAR). We previously reported that serum-affected keratinocyte differentiation and function; namely, it inhibited keratinization, decreased loricrin (LOR) and claudin (CLDN) 1 expression, increased keratin (K) 4 and CLDN4 levels, and reduced paracellular permeability in three-dimensional (3D) cultures of mouse keratinocytes (COCA). Contrarily, RAR inhibition reversed these changes. Here, we aimed to examine whether atRA exerted the same effects as serum, and whether it was involved in the differential oral mucosa keratinization among animal species. Porcine oral mucosal keratinocytes, which form non-keratinized epithelium in vivo, established keratinized epithelium in 3D cultures. Both mouse and porcine sera induced non-keratinized epithelium at 0.1% in COCA 3D cultures. Although atRA caused the same changes as serum, its effective concentration differed. atRA inhibited keratinization at 0.1 nM and 1 nM in porcine or human keratinocytes and COCA, respectively. Furthermore, atRA upregulated CLDN7 in the cytoplasm but not in cell-cell contacts. These atRA-induced changes were reverted by RAR inhibition. The results indicate that serum-induced changes are probably due to the effect of serum-derived atRA, and that mouse keratinocytes require higher atRA concentrations to suppress keratinization than porcine and human keratinocytes. We propose that the lower susceptibility of mouse keratinocytes to atRA, rather than a lower retinol concentration, is a possible reason for the keratinization of mouse oral mucosal epithelium.
Assuntos
Epitélio/efeitos dos fármacos , Mucosa Esofágica/efeitos dos fármacos , Queratinócitos/efeitos dos fármacos , Queratinas/metabolismo , Mucosa Bucal/efeitos dos fármacos , Tretinoína/farmacologia , Animais , Benzoatos/farmacologia , Células Cultivadas , Epitélio/metabolismo , Mucosa Esofágica/metabolismo , Humanos , Queratinócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Mucosa Bucal/metabolismo , Estilbenos/farmacologia , Suínos , Tretinoína/antagonistas & inibidoresRESUMO
We isolated the transmembrane and coiled-coil domains 5A (Tmco5A) gene using polymerase chain reaction-based subtraction technique and showed that Tmco5A was predominantly expressed in rat testes starting at 4 weeks of postnatal development. When expressed in COS7 cells, TMCO5A was found to be distributed in the endoplasmic reticulum-nuclear membrane (ER-NM) of cells as a membrane-associated protein, while TMCO5AΔC lacking the transmembrane region (TM) mislocalized and diffused throughout the cytoplasm. The result suggested that TM is responsible for the retention of TMCO5A at the ER-NM. Immunocytochemical and immunoblotting analyses indicated that TMCO5A was localized along the posterior part of the nuclei in both round and elongated rat spermatids but disappeared from epididymal spermatozoa. Double immunolabeling of isolated spermatids with the anti-TMCO5A and the anti-ß tubulin antibodies showed that TMCO5A was always found to be closely associated with developing manchette microtubules but did not completely colocalize with them. On the other hand, we found that almost all TMCO5A colocalized with SUN4, a linker of nucleoskeleton and cytoskeleton complex protein present at the posterior part of spermatid nuclei. These data suggested that TMCO5A is located closer to the nuclei than the manchette microtubules. It is likely that TMCO5A, in association with manchette microtubules, is involved in the process of spermiogenesis.
Assuntos
Proteínas de Membrana/metabolismo , Microtúbulos/metabolismo , Membrana Nuclear/metabolismo , Espermátides/crescimento & desenvolvimento , Espermatogênese/fisiologia , Sequência de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Retículo Endoplasmático/metabolismo , Imuno-Histoquímica , Masculino , Proteínas Nucleares/metabolismo , Domínios Proteicos/fisiologia , Ratos , Ratos Wistar , Musaranhos , Testículo/metabolismoRESUMO
Nisin is a food preservative produced by Lactococcus lactis subsp. lactis. Previous blood biochemical research revealed that nisin has physiological effects in mammals; although the site of action has yet to be identified, keratinocytes have been proposed as a possible target. In this study, we investigated whether nisin affects keratinocytes by examining the effects on eukaryotic intermediate filaments in HaCaT human keratinocytes. Treatment with 93 µg/ml nisin for 24 h decreased the localization of the intermediate filament proteins cytokeratin (CK)5 and CK17 at the cell periphery, which were distributed in a limited area in a ring- or net-like shape. However, this was not observed upon treatment for 6 h. The results of a serial dilution assay revealed that the effect on CK17 localization depends on the nisin concentration and were observed at ≥47 µg/ml. Moreover, this effect was partially blocked by treatment with the calcium channel blocker bepridil. Thus, despite the long history of nisin as being safe for humans, it has measurable effects on the keratinocyte cytoskeleton. Our findings also indicate that CK5 and CK17 can serve as markers for evaluating the effects of nisin on keratinocytes.
Assuntos
Conservantes de Alimentos/farmacologia , Proteínas de Filamentos Intermediários/metabolismo , Queratinócitos/metabolismo , Nisina/farmacologia , Bepridil/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Células Cultivadas , Citoesqueleto/metabolismo , Relação Dose-Resposta a Droga , Humanos , Queratina-17/metabolismo , Queratina-5/metabolismo , Queratinócitos/citologia , Lactococcus lactis/metabolismo , Nisina/antagonistas & inibidores , Nisina/biossínteseRESUMO
Vitamin A, which is found in serum, is known to affect keratinocyte proliferation, epidermal differentiation, and keratinization. In mice, stratified epithelia in the oral cavity, esophagus, and forestomach are keratinized; however, these epithelia are not keratinized in humans. Several studies have reported that three-dimensional (3D) cultures of human keratinocytes in serum-containing medium could form keratinized epithelia. Here, we evaluated the effects of serum on the morphology, expression, and localization of differentiation markers and tight junction proteins, and paracellular permeability in 3D cultures of mouse keratinocytes. We found that only 0.1% calcium-depleted serum inhibited keratinization and induced a change in the expression of differentiation marker proteins from loricrin to keratin 4; the inhibition of retinoic acid receptor-mediated signaling reversed these changes. Furthermore, the serum reduced claudin-1 protein expression and prevented its localization at occludin-positive spots on the surface of 3D cultures. On the other hand, the serum increased the protein expression of claudin-4, occludin, zonula occludens-1, and E-cadherin. These changes may contribute to the reduction of the transepithelial electrical resistance by approximately half. In conclusion, mouse keratinocytes derived from the epidermis formed non-keratinized structures in 3D cultures in response to vitamin A in serum. The results suggest that retinoic acid receptor-mediated signaling may be inhibited in the mouse epithelia in the oral cavity, esophagus, and forestomach as well as the epidermis, leading to the keratinization of these epithelia.
Assuntos
Técnicas de Cultura de Células/métodos , Queratinócitos/citologia , Queratinas/metabolismo , Receptores do Ácido Retinoico/metabolismo , Transdução de Sinais , Proteínas de Junções Íntimas/metabolismo , Junções Íntimas/metabolismo , Animais , Biomarcadores/sangue , Diferenciação Celular , Linhagem Celular , Queratinócitos/metabolismo , Queratinas/sangue , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Microscopia de Fluorescência , Proteínas de Junções Íntimas/sangue , Junções Íntimas/químicaRESUMO
Keratinocytes in the oral mucosal epithelium, which is a non-keratinized stratified epithelium, are exposed to various stimuli from the oral cavity. JNK and p38 are stress-activated mitogen-activated protein kinases (MAPKs) that are phosphorylated by various stimuli and are involved in the assembly and disassembly of tight junctions (TJs) in keratinocytes. Therefore, we investigated the effects of stress-activated MAPKs on TJs in a mouse keratinocyte cell line during cell-cell junction formation in two-dimensional (2D) cultures or stratification to form non-keratinized epithelium in 3D cultures. In 2D cultures, calcium induced zipper-like staining for ZO-1 at 2 h and string-like staining for ZO-1 at 12 h, which indicated immature and mature cell-cell junctions, respectively. Anisomycin (AM), a JNK and p38 activator, inhibited formation of string-like staining for ZO-1, whereas inhibition of JNK, but not p38, after AM treatment restored string-like staining for ZO-1, although claudins (CLDNs) 4, 6, and 7 did not completely colocalize to ZO-1-positive sites. In 3D cultures, AM treatment for 2 weeks activated only p38, suppressed flattening of the superficial cells, removed CLDN7 from ZO-1-positive spots on the surface of 3D cultures, which represent TJs, and decreased transepithelial electrical resistance. Thus, short-term AM treatment inhibited maturation of cell-cell junctions by JNK, but not p38, activation. p38 activation by long-term AM treatment affected morphology of stratified structures and paracellular permeability, which was increased by CLDN7 removal from TJs. Various chronic stimuli that activate stress-activated MAPKs may weaken the keratinocyte barrier and be involved in TJ-related diseases.
Assuntos
Anisomicina/farmacologia , Técnicas de Cultura de Células , Permeabilidade da Membrana Celular/efeitos dos fármacos , Claudinas/biossíntese , Junções Intercelulares/efeitos dos fármacos , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Animais , Células Cultivadas , Claudinas/metabolismo , Junções Intercelulares/metabolismo , CamundongosRESUMO
The uncarboxylated form of osteocalcin (GluOC) regulates glucose and lipid metabolism in mice. We previously showed that low-dose (≤10 ng/ml) GluOC induces the expression of adiponectin and peroxisome proliferator-activated receptor γ (PPARγ) via a cAMP-PKA-ERK-CREB signaling pathway in 3T3-L1 adipocytes. We also noticed that high-dose (≥20 ng/ml) GluOC inhibits the expression of adiponectin and PPARγ in these cells. We have here explored the mechanism underlying these effects of high-dose GluOC. High-dose GluOC triggered morphological changes in 3T3-L1 adipocytes suggestive of the induction of cell death. It activated the putative GluOC receptor GPRC6A and thereby induced the production of cAMP and activation of protein kinase A (PKA), similar to signaling by low-dose GluOC with the exception that the catalytic subunit of PKA also entered the nucleus. Cytosolic PKA induced phosphorylation of cAMP response element-binding protein (CREB) at serine-133 via extracellular signal-regulated kinase (ERK). Nuclear PKA appeared to mediate the inhibitory phosphorylation of salt-inducible kinase 2 (SIK2) at serine-358 and thereby to alleviate the inhibitory phosphorylation of the CREB co-activator p300 at serine-89. The activation of CREB and p300 resulted in increased expression of the transcription factor FoxO1 and consequent upregulation of Fas ligand (FasL) at the plasma membrane. The interaction of FasL with Fas on neighboring adipocytes triggered the phosphorylation at threonine-357/serine-358 and homotrimerization of mixed-lineage kinase domain-like protein (MLKL), a key regulator of necroptosis, as well as Ca2+ influx via transient receptor potential melastatin 7 (TRPM7), the generation of reactive oxygen species and lipid peroxides, and dephosphorylation of dynamin-related protein 1 (DRP1) at serine-637, resulting in mitochondrial fragmentation. Together, our results indicate that high-dose GluOC triggers necroptosis through upregulation of FasL at the plasma membrane in a manner dependent of activation of CREB-p300, followed by the activation of Fas signaling in neighboring adipocytes.
Assuntos
Morte Celular/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína Ligante Fas/genética , Receptor fas/genética , Fatores de Transcrição de p300-CBP/genética , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Adiponectina/genética , Animais , Morte Celular/efeitos dos fármacos , Membrana Celular/genética , AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/genética , Dinaminas/genética , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Glucose/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Osteocalcina/farmacologia , Fosforilação/efeitos dos fármacos , Canais de Cátion TRPM/genéticaRESUMO
Tight junctions (TJs) are essential cell adhesion structures that act as a barrier to separate the internal milieu from the external environment in multicellular organisms. Although their major constituents have been identified, it is unknown how the formation of TJs is regulated. TJ formation depends on the preceding formation of adherens junctions (AJs) in epithelial cells; however, the underlying mechanism remains to be elucidated. In this study, loss of AJs in α-catenin-knockout (KO) EpH4 epithelial cells altered the lipid composition of the plasma membrane (PM) and led to endocytosis of claudins, a major component of TJs. Sphingomyelin with long-chain fatty acids and cholesterol were enriched in the TJ-containing PM fraction. Depletion of cholesterol abolished the formation of TJs. Conversely, addition of cholesterol restored TJ formation in α-catenin-KO cells. Collectively, we propose that AJs mediate the formation of TJs by increasing the level of cholesterol in the PM.
Assuntos
Junções Aderentes/genética , Lipídeos de Membrana/metabolismo , Esfingomielinas/genética , alfa Catenina/genética , Junções Aderentes/metabolismo , Adesão Celular/genética , Linhagem Celular , Colesterol/genética , Colesterol/metabolismo , Endocitose/genética , Células Epiteliais/metabolismo , Técnicas de Inativação de Genes , Humanos , Lipídeos de Membrana/genética , Esfingomielinas/química , Junções Íntimas/genéticaRESUMO
Molecular investigations were performed in order to determine the major characteristics of voltage-gated Na+ channel ß-subunits in mouse vas deferens. The use of real-time quantitative PCR showed that the expression of Scn1b was significantly higher than that of other ß-subunit genes (Scn2b - Scn4b). Immunoreactivity of Scn1b proteins was also detected in the inner circular and outer longitudinal smooth muscle of mouse vas deferens. In whole-cell recordings, the actions of 4,9-anhydroTTX on voltage-gated Na+ current peak amplitude in myocytes (i.e., native INa) were compared with its inhibitory potency on recombinant NaV1.6 channels (expressed in HEK293 cells). A depolarizing rectangular voltage-pulse elicited a fast and transient inward native INa and recombinant NaV1.6 expressed in HEK293 cells (i.e., recombinant INa). The current decay of native INa was similar to the recombinant NaV1.6 current co-expressed with ß1-subunits. The current-voltage (I-V) relationships of native INa were similar to those of recombinant NaV1.6 currents co-expressed with ß1-subunits. Application of 4,9-anhydroTTX inhibited the peak amplitude of native INa (K i = 510 nM), recombinant INa (K i = 112 nM), and recombinant INa co-expressed with ß1-subunits (K i = 92 nM). The half-maximal (Vhalf) activation and inactivation of native INa values were similar to those observed in recombinant INa co-expressed with ß1-subunits. These results suggest that ß1-subunit proteins are likely to be expressed mainly in the smooth muscle layers of murine vas deferens and that 4,9-anhydroTTX inhibited not only native INa but also recombinant INa and recombinant INa co-expressed with ß1-subunits in a concentration-dependent manner.
Assuntos
Miócitos de Músculo Liso/efeitos dos fármacos , Subunidades Proteicas/fisiologia , Bloqueadores dos Canais de Sódio/farmacologia , Tetrodotoxina/análogos & derivados , Ducto Deferente/citologia , Canais de Sódio Disparados por Voltagem/fisiologia , Animais , Cérebro/efeitos dos fármacos , Cérebro/fisiologia , Células HEK293 , Humanos , Masculino , Camundongos Endogâmicos BALB C , Miócitos de Músculo Liso/fisiologia , Proteínas Recombinantes , Tetrodotoxina/farmacologiaRESUMO
Intestinal epithelial cells are the first targets of ingested mycotoxins, such as ochratoxin A, citrinin and deoxynivalenol. It has been reported that paracellular permeability regulated by tight junctions is modulated by several mycotoxins by reducing the expression of specific claudins and integral membrane proteins in cell-cell contacts, accompanied by increase in phosphorylation of mitogen-activated protein kinases, including extracellular signal-related kinase (ERK) 1/2, p38 and c-Jun NH2-terminal protein kinase. Claudin-2 is expressed in the deep crypt cells, but not in the villus/surface cells in vivo. While Caco-2, T84 and IPEC-J2 cells, which are widely used intestinal epithelial cell lines to assess the influence of mycotoxins, do not express claudin-2, CMT93-II cells express claudin-2. We previously reported that inhibition of the ERK pathway reduced claudin-2 levels in cell-cell contacts in CMT93-II cells. In this study, we examined whether ochratoxin A, citrinin and deoxynivalenol affect claudin-2 expression and ERK1/2 phosphorylation in CMT93-II cells. We found that all mycotoxins reduced claudin-2 expression in cell-cell contacts, with reduction (by citrinin and deoxynivalenol) or no change (by ochratoxin A) in phosphorylated ERK1/2. All mycotoxins increased transepithelial electrical resistance, but did not affect flux of fluorescein. While ochratoxin A and citrinin are known to be nephrotoxic, only deoxynivalenol reduced claudin-2 expression in MDCK II cells derived from the renal tubule. These results suggest that claudin-2 expression is regulated not only by the ERK pathway, but also by other pathways in an organ-specific manner.
Assuntos
Citrinina/toxicidade , Claudina-2/biossíntese , Células Epiteliais/patologia , Expressão Gênica/efeitos dos fármacos , Ocratoxinas/toxicidade , Tricotecenos/toxicidade , Animais , Aspergillus ochraceus/patogenicidade , Butadienos/farmacologia , Células CACO-2 , Linhagem Celular , Cães , Inibidores Enzimáticos/farmacologia , Fusarium/patogenicidade , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Mucosa Intestinal/patologia , Células Madin Darby de Rim Canino , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Nitrilas/farmacologia , Penicillium/patogenicidade , Permeabilidade/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Reto/citologia , Reto/metabolismoRESUMO
Tektins (TEKTs) are filamentous proteins associated with microtubules in cilia, flagella, basal bodies, and centrioles. Five TEKTs (TEKT1, -2, -3, -4, and -5) have been identified as components of mammalian sperm flagella. We previously reported that TKET1 and -3 are also present in the heads of rodent spermatozoa. The present study clearly demonstrates that TEKT2 is present at the acrosome cap whereas TEKT3 resides just beneath the plasma membrane of the post-acrosomal region of sperm heads in unactivated bull spermatozoa, and builds on the distributional differences of TEKT1, -2, and -3 on sperm heads. We also discovered that hyperactivation of bull spermatozoa by cell-permeable cAMP and calyculin A, a protein phosphatase inhibitor, promoted translocation of TEKT3 from the post-acrosomal region to the equatorial segment in sperm heads, and that TEKT3 accumulated at the equatorial segment is lost upon acrosome reaction. Thus, translocation of TEKT3 to the equatorial segment may be a capacitation- or hyperactivation-associated phenomenon in bull spermatozoa. Mol. Reprod. Dev. 84: 30-43, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
Acrossomo/metabolismo , Bucladesina/farmacologia , Proteínas dos Microtúbulos/metabolismo , Oxazóis/farmacologia , Reação Acrossômica/efeitos dos fármacos , Animais , Bovinos , Masculino , Toxinas Marinhas , Transporte Proteico/efeitos dos fármacosRESUMO
Three-dimensional (3D) cell culture is a powerful in vitro technique to study the stratification and differentiation of keratinocytes. However, culture conditions, including culture media, supplements, and scaffolds (e.g., collagen gels with or without fibroblasts), can vary considerably. Here, we evaluated the roles of calcium, L-ascorbic acid phosphate magnesium salt n-hydrate (APM), and keratinocyte growth factor (KGF) in a chemically defined medium, EpiLife, in 3D cultures of primary human epidermal keratinocytes directly plated on polycarbonate filter inserts under airlifted or submerged conditions. Eight culture media containing various combinations of these three supplements were examined. Calcium was necessary for the stratification and differentiation of keratinocytes based on the localization of keratins and involucrin. However, the localization patterns of keratins and integrin ß4 were partially disrupted and Ki67-positive basal cells almost disappeared 3 weeks after airlift. The addition of KGF, but not APM, prevented these changes. Further addition of APM markedly improved the tissue architecture, including basal cell morphology and the appearance of keratohyalin granules and localized involucrin in the upper suprabasal cells, even after 1 week. Although the submerged culture also formed cornified epithelium-like multilayers, involucrin was localized in the cornified layer, where nuclei were often found. Based on these results, it is most effective to culture keratinocytes at the air-liquid interface in EpiLife medium supplemented with calcium, APM, and KGF to form well-organized and orthokeratinized multilayers as skin analogues.
Assuntos
Ácido Ascórbico/farmacologia , Cálcio/farmacologia , Meios de Cultura/química , Meios de Cultura/farmacologia , Fator 7 de Crescimento de Fibroblastos/farmacologia , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Adulto , Células Cultivadas , Humanos , Queratinócitos/metabolismoRESUMO
Epithelial cells define the boundary between the outside and the inside of our body by constructing the diffusion barrier. Tight junctions (TJs) of epithelial cells function as barriers against invasion of harmful microorganisms into the human body and free diffusion of water or ions from the body. Therefore, formation of TJs has to be strictly controlled in epithelial cells. However, the molecular mechanisms governing this regulation are largely unknown. In this study, we identified Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) as a regulator of the barrier function of TJs. CaMKII inhibition led to enlargement of TJ-areas and up-regulation of the barrier function. CaMKII inhibition induced excess TJ formation in part by the activation of AMP-activated protein kinase (AMPK) and subsequent phosphorylation of claudin-1. As up-regulation of epithelial barriers is essential for the prevention of chronic inflammatory diseases, the identification of CaMKII as a modulator of TJ function paves the way for the development of new drugs to treat these diseases.