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1.
J Cell Sci ; 135(5)2022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-34156466

RESUMO

Store-operated Ca2+ entry is a central component of intracellular Ca2+ signaling pathways. The Ca2+ release-activated channel (CRAC) mediates store-operated Ca2+ entry in many different cell types. The CRAC channel is composed of the plasma membrane (PM)-localized Orai1 channel and endoplasmic reticulum (ER)-localized STIM1 Ca2+ sensor. Upon ER Ca2+ store depletion, Orai1 and STIM1 form complexes at ER-PM junctions, leading to the formation of activated CRAC channels. Although the importance of CRAC channels is well described, the underlying mechanisms that regulate the recruitment of Orai1 to ER-PM junctions are not fully understood. Here, we describe the rapid and transient S-acylation of Orai1. Using biochemical approaches, we show that Orai1 is rapidly S-acylated at cysteine 143 upon ER Ca2+ store depletion. Importantly, S-acylation of cysteine 143 is required for Orai1-mediated Ca2+ entry and recruitment to STIM1 puncta. We conclude that store depletion-induced S-acylation of Orai1 is necessary for recruitment to ER-PM junctions, subsequent binding to STIM1 and channel activation.


Assuntos
Canais de Cálcio , Cálcio , Acilação , Cálcio/metabolismo , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Sinalização do Cálcio , Membrana Celular/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteína ORAI1/genética , Proteína ORAI1/metabolismo , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/metabolismo
2.
Biomed Pharmacother ; 139: 111615, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34243598

RESUMO

BACKGROUND: Severe acidosis deteriorates cardiac injury. Rat coronary arteries (RCAs) are unusually hypercontractive to extracellular (o) acidosis (EA). TMEM16A-encoded anoctamin 1 (ANO1), a Ca2+-activated chloride channel (CaCC), plays an important role in regulating coronary arterial tension. PURPOSE: We tested the possibility that the activation of CaCCs in the arterial smooth muscle cell (ASMC) contributes to EA-induced RCA constriction. METHODS: ANO1 expression was detected with immunofluorescence staining and Western blot. TMEM16A mRNA was assessed with quantitative Real-Time PCR. Cl- currents and membrane potentials were quantified with a patch clamp. The vascular tension was recorded with a myograph. Intracellular (i) level of Cl- and Ca2+ was measured with fluorescent molecular probes. RESULTS: ANO1 was expressed in all tested arterial myocytes, but was much more abundant in RCA ASMCs as compared with ASMCs isolated from rat cerebral basilar, intrarenal and mesenteric arteries. EA reduced [Cl-]i levels, augmented CaCC currents exclusively in RCA ASMCs and depolarized RCA ASMCs to a greater extent. Cl- deprivation, which depleted [Cl-]i by incubating the arteries or their ASMCs in Cl--free bath solution, decreased EA-induced [Cl-]i reduction, diminished EA-induced CaCC augmentation and time-dependently depressed EA-induced RCA constriction. Inhibitor studies showed that these EA-induced effects including RCA constriction, CaCC current augmentation, [Cl-]i reduction and/or [Ca2+]i elevation were depressed by various Cl- channel blockers, [Ca2+]i release inhibitors and L-type voltage-gated Ca2+ channel inhibitor nifedipine. ANO1 antibody attenuated all observed changes induced by EA in RCA ASMCs. CONCLUSION: The greater activity of RCA ASMC CaCCs complicated with an enhanced Ca2+ mobilization from both [Ca2+]i release and [Ca2+]o influx plays a pivotal role in the distinctive hypercontractility of RCAs to acidosis. Translation of these findings to human beings may lead to a new conception in our understanding and treating cardiac complications in severe acidosis.


Assuntos
Acidose/metabolismo , Anoctamina-1/metabolismo , Vasos Coronários/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Vasoconstrição/fisiologia , Acidose/tratamento farmacológico , Animais , Cálcio/metabolismo , Canais de Cloreto/metabolismo , Cloretos/metabolismo , Vasos Coronários/efeitos dos fármacos , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Artérias Mesentéricas/efeitos dos fármacos , Artérias Mesentéricas/metabolismo , Células Musculares/efeitos dos fármacos , Células Musculares/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Nifedipino/farmacologia , Técnicas de Patch-Clamp/métodos , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Vasoconstrição/efeitos dos fármacos
3.
FASEB J ; 35(8): e21756, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34270805

RESUMO

Protein tyrosine phosphatase non-receptor type 5 (PTPN5), also called striatal-enriched protein tyrosine phosphatase (STEP), is highly expressed in neurons of the basal ganglia, hippocampus, cortex, and related structures, also in the pituitary. Gonadotropins are the key regulator of the reproduction in mammals. In this study, PTPN5 is detected to express in murine pituitary in a developmental manner. Moreover, the expression of PTPN5 in the pituitary is heavily reduced after ovary removal. Follicle-stimulating hormone (FSH) secretion in gonadotropes is regulated by PTPN5 via binding GnRH to GnRH-R. Two parallel signaling pathways, Gs-protein kinase A (PKA)-PTPN5 and Gq-phospholipases C (PLC)-p38 MAPK-PTPN5, cooperatively regulate GnRH-induced FSH secretion. We also show that influx of Ca2+ activates the Ca2+ -dependent phosphatase calcineurin, leading to the phosphorylation and activation of PTPN5. The intracellular release of Ca2+ is reduced via TC2153. In conclusion, blocking or knocking out of PTPN5 reduces the release of FSH in whole pituitary. Mechanically, PTPN5 regulates gonadotropes' function through regulating intracellular calcium homeostasis.


Assuntos
Cálcio/metabolismo , Hormônio Foliculoestimulante/metabolismo , Homeostase , Sistema de Sinalização das MAP Quinases , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Feminino , Hormônio Foliculoestimulante/genética , Masculino , Camundongos , Camundongos Knockout , Proteínas Tirosina Fosfatases não Receptoras/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genética
4.
Int J Mol Sci ; 22(13)2021 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-34198993

RESUMO

Mechanistic target of rapamycin complex 1 (mTORC1) is a master growth regulator by controlling protein synthesis and autophagy in response to environmental cues. Amino acids, especially leucine and arginine, are known to be important activators of mTORC1 and to promote lysosomal translocation of mTORC1, where mTORC1 is thought to make contact with its activator Rheb GTPase. Although amino acids are believed to exclusively regulate lysosomal translocation of mTORC1 by Rag GTPases, how amino acids increase mTORC1 activity besides regulation of mTORC1 subcellular localization remains largely unclear. Here we report that amino acids also converge on regulation of the TSC2-Rheb GTPase axis via Ca2+/calmodulin (CaM). We showed that the amino acid-mediated increase of intracellular Ca2+ is important for mTORC1 activation and thereby contributes to the promotion of nascent protein synthesis. We found that Ca2+/CaM interacted with TSC2 at its GTPase activating protein (GAP) domain and that a CaM inhibitor reduced binding of CaM with TSC2. The inhibitory effect of a CaM inhibitor on mTORC1 activity was prevented by loss of TSC2 or by an active mutant of Rheb GTPase, suggesting that a CaM inhibitor acts through the TSC2-Rheb axis to inhibit mTORC1 activity. Taken together, in response to amino acids, Ca2+/CaM-mediated regulation of the TSC2-Rheb axis contributes to proper mTORC1 activation, in addition to the well-known lysosomal translocation of mTORC1 by Rag GTPases.


Assuntos
Aminoácidos/metabolismo , Sinalização do Cálcio , Cálcio/metabolismo , Calmodulina/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteína Enriquecida em Homólogo de Ras do Encéfalo/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa/metabolismo , Linhagem Celular , Técnicas de Silenciamento de Genes , Humanos , Espaço Intracelular/metabolismo , Lisossomos/metabolismo , Modelos Biológicos , Ligação Proteica , Transdução de Sinais
5.
Int J Mol Sci ; 22(12)2021 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-34201319

RESUMO

Proper skeletal muscle function is controlled by intracellular Ca2+ concentration and by efficient production of energy (ATP), which, in turn, depend on: (a) the release and re-uptake of Ca2+ from sarcoplasmic-reticulum (SR) during excitation-contraction (EC) coupling, which controls the contraction and relaxation of sarcomeres; (b) the uptake of Ca2+ into the mitochondrial matrix, which stimulates aerobic ATP production; and finally (c) the entry of Ca2+ from the extracellular space via store-operated Ca2+ entry (SOCE), a mechanism that is important to limit/delay muscle fatigue. Abnormalities in Ca2+ handling underlie many physio-pathological conditions, including dysfunction in ageing. The specific focus of this review is to discuss the importance of the proper architecture of organelles and membrane systems involved in the mechanisms introduced above for the correct skeletal muscle function. We reviewed the existing literature about EC coupling, mitochondrial Ca2+ uptake, SOCE and about the structural membranes and organelles deputed to those functions and finally, we summarized the data collected in different, but complementary, projects studying changes caused by denervation and ageing to the structure and positioning of those organelles: a. denervation of muscle fibers-an event that contributes, to some degree, to muscle loss in ageing (known as sarcopenia)-causes misplacement and damage: (i) of membrane structures involved in EC coupling (calcium release units, CRUs) and (ii) of the mitochondrial network; b. sedentary ageing causes partial disarray/damage of CRUs and of calcium entry units (CEUs, structures involved in SOCE) and loss/misplacement of mitochondria; c. functional electrical stimulation (FES) and regular exercise promote the rescue/maintenance of the proper architecture of CRUs, CEUs, and of mitochondria in both denervation and ageing. All these structural changes were accompanied by related functional changes, i.e., loss/decay in function caused by denervation and ageing, and improved function following FES or exercise. These data suggest that the integrity and proper disposition of intracellular organelles deputed to Ca2+ handling and aerobic generation of ATP is challenged by inactivity (or reduced activity); modifications in the architecture of these intracellular membrane systems may contribute to muscle dysfunction in ageing and sarcopenia.


Assuntos
Trifosfato de Adenosina/metabolismo , Envelhecimento/patologia , Cálcio/metabolismo , Músculo Esquelético/patologia , Doenças Musculares/patologia , Organelas/patologia , Envelhecimento/metabolismo , Animais , Humanos , Músculo Esquelético/metabolismo , Doenças Musculares/metabolismo , Organelas/metabolismo
6.
Int J Mol Sci ; 22(13)2021 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-34202585

RESUMO

Cultured keratinocytes are desirable models for biological and medical studies. However, primary keratinocytes are difficult to maintain, and there has been little research on lingual keratinocyte culture. Here, we investigated the effect of Y-27632, a Rho kinase (ROCK) inhibitor, on the immortalization and characterization of cultured rat lingual keratinocyte (RLKs). Three Y-27632-supplemented media were screened for the cultivation of RLKs isolated from Sprague-Dawley rats. Phalloidin staining and TUNEL assay were applied to visualize cytoskeleton dynamics and cell apoptosis following Y-27632 removal. Label-free proteomics, RT-PCR, calcium imaging, and cytogenetic studies were conducted to characterize the cultured cells. Results showed that RLKs could be conditionally immortalized in a high-calcium medium in the absence of feeder cells, although they did not exhibit normal karyotypes. The removal of Y-27632 from the culture medium led to reversible cytoskeletal reorganization and nuclear enlargement without triggering apoptosis, and a total of 239 differentially expressed proteins were identified by proteomic analysis. Notably, RLKs derived from the non-taste epithelium expressed some molecular markers characteristic of taste bud cells, yet calcium imaging revealed that they rarely responded to tastants. Collectively, we established a high-calcium and feeder-free culture method for the long-term maintenance of RLKs. Our results shed some new light on the immortalization and differentiation of lingual keratinocytes.


Assuntos
Amidas/farmacologia , Cálcio/metabolismo , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Animais , Técnicas de Cultura de Células , Células Cultivadas , Ratos
7.
Int J Mol Sci ; 22(11)2021 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-34204139

RESUMO

The prohibitin (PHB)-binding compound fluorizoline as well as PHB-downregulation activate the integrated stress response (ISR) in HEK293T and U2OS human cell lines. This activation is denoted by phosphorylation of eIF2α and increases in ATF4, ATF3, and CHOP protein levels. The blockage of the activation of the ISR by overexpression of GRP78, as well as an increase in IRE1 activity, indicate the presence of ER stress after fluorizoline treatment. The inhibition of the ER stress response in HEK293T and U2OS led to increased sensitivity to fluorizoline-induced apoptosis, indicating a pro-survival role of this pathway after fluorizoline treatment in these cell lines. Fluorizoline induced an increase in calcium concentration in the cytosol and the mitochondria. Finally, two different calcium chelators reduced fluorizoline-induced apoptosis in U2OS cells. Thus, we have found that fluorizoline causes increased ER stress and activation of the integrated stress response, which in HEK293T and U2OS cells are protective against fluorizoline-induced apoptosis.


Assuntos
Apoptose , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Tiazóis/farmacologia , Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Linhagem Celular Tumoral , Respiração Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Células HEK293 , Homeostase/efeitos dos fármacos , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais/efeitos dos fármacos
8.
Int J Mol Sci ; 22(12)2021 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-34204776

RESUMO

Point mutations in the genes encoding the skeletal muscle isoforms of tropomyosin can cause a range of muscle diseases. The amino acid substitution of Arg for Pro residue in the 90th position (R90P) in γ-tropomyosin (Tpm3.12) is associated with congenital fiber type disproportion and muscle weakness. The molecular mechanisms underlying muscle dysfunction in this disease remain unclear. Here, we observed that this mutation causes an abnormally high Ca2+-sensitivity of myofilaments in vitro and in muscle fibers. To determine the critical conformational changes that myosin, actin, and tropomyosin undergo during the ATPase cycle and the alterations in these changes caused by R90P replacement in Tpm3.12, we used polarized fluorimetry. It was shown that the R90P mutation inhibits the ability of tropomyosin to shift towards the outer domains of actin, which is accompanied by the almost complete depression of troponin's ability to switch actin monomers off and to reduce the amount of the myosin heads weakly bound to F-actin at a low Ca2+. These changes in the behavior of tropomyosin and the troponin-tropomyosin complex, as well as in the balance of strongly and weakly bound myosin heads in the ATPase cycle may underlie the occurrence of both abnormally high Ca2+-sensitivity and muscle weakness. BDM, an inhibitor of myosin ATPase activity, and W7, a troponin C antagonist, restore the ability of tropomyosin for Ca2+-dependent movement and the ability of the troponin-tropomyosin complex to switch actin monomers off, demonstrating a weakening of the damaging effect of the R90P mutation on muscle contractility.


Assuntos
Contração Muscular/genética , Mutação/genética , Oximas/farmacologia , Sulfonamidas/farmacologia , Tropomiosina/genética , Actinas/metabolismo , Animais , Cálcio/metabolismo , Contração Muscular/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Miofibrilas/efeitos dos fármacos , Miofibrilas/metabolismo , Miosinas/metabolismo , Coelhos , Troponina/metabolismo
9.
Int J Mol Sci ; 22(11)2021 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-34205207

RESUMO

Since dysregulation of intracellular calcium (Ca2+) levels is a common occurrence in neurodegenerative diseases, including Alzheimer's disease (AD), the study of proteins that can correct neuronal Ca2+ dysregulation is of great interest. In previous work, we have shown that plasma membrane Ca2+-ATPase (PMCA), a high-affinity Ca2+ pump, is functionally impaired in AD and is inhibited by amyloid-ß peptide (Aß) and tau, two key components of pathological AD hallmarks. On the other hand, sorcin is a Ca2+-binding protein highly expressed in the brain, although its mechanism of action is far from being clear. Sorcin has been shown to interact with the intracellular sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), and other modulators of intracellular Ca2+ signaling, such as the ryanodine receptor or presenilin 2, which is closely associated with AD. The present work focuses on sorcin in search of new regulators of PMCA and antagonists of Aß and tau toxicity. Results show sorcin as an activator of PMCA, which also prevents the inhibitory effects of Aß and tau on the pump, and counteracts the neurotoxicity of Aß and tau by interacting with them.


Assuntos
Doença de Alzheimer/genética , Proteínas de Ligação ao Cálcio/genética , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/genética , Encéfalo/metabolismo , Encéfalo/patologia , Cálcio/metabolismo , Sinalização do Cálcio/genética , Humanos , Neurônios/metabolismo , Neurônios/patologia , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Presenilina-2/genética , Ligação Proteica/genética , Mapas de Interação de Proteínas/genética , Proteínas tau/genética
10.
Int J Mol Sci ; 22(11)2021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-34206144

RESUMO

The Casparian strip domain protein 1 (OsCASP1) is necessary for the formation of the Casparian strip (CS) in the rice endodermis. It also controls Ca2+ transport to the stele. Here, we demonstrated that OsCASP1 overexpression enhanced Ca tolerance in rice. Under normal conditions, OsCASP1-overexpressed lines showed similar concentrations of essential metals in the roots and shoots compared to the wild type, while under high Ca conditions, Ca in the roots, shoots, and xylem sap of the OsCASP1-overexpressed lines was significantly decreased. This did not apply to other essential metals. Ca-inhibited growth was significantly alleviated in the OsCASP1-overexpressed lines. Furthermore, OsCASP1 overexpression resulted in earlier formation of both the CS and functional apoplastic barrier in the endodermis but did not induce ectopic CS formation in non-endodermal cell layers and affect suberin accumulation in the endodermis. These results indicate that the overexpression of OsCASP1 promotes CS formation in endodermal cells and inhibits Ca2+ transport by the apoplastic pathway, restricting Ca accumulation in the roots and shoots under high Ca conditions. Taken together, the results suggest that OsCASP1 overexpression is an effective way to improve rice adaptation to high Ca environments.


Assuntos
Cálcio/metabolismo , Caspase 1/genética , Oryza/genética , Caspase 1/metabolismo , Parede Celular/genética , Regulação da Expressão Gênica de Plantas/genética , Oryza/crescimento & desenvolvimento , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento
11.
Int J Mol Sci ; 22(12)2021 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-34207344

RESUMO

Osteoporosis is a chronic disease characterized by low bone mass caused by increased bone turnover and impaired bone microarchitecture. In treatment, we use antiresorptive or anabolic drugs, which usually have a unidirectional effect, i.e., they inhibit the activity of osteoclasts or stimulate the effect of osteoblasts. Strontium ranelate is an anti-osteoporosis drug with a unique mechanism of action (used primarily in postmenopausal women). Unlike other medicines, it has a multidirectional effect on bone tissue, intensifying osteoblastogenesis while inhibiting osteoclastogenesis. It turns out that this effect is demonstrated by strontium ions, an element showing physical and chemical similarity to calcium, the basic element that builds the mineral fraction of bone. As a result, strontium acts through the calcium-sensing receptor (CaSR) receptor in bone tissue cells. In recent years, there has been a significant increase in interest in the introduction of strontium ions in place of calcium ions in ceramics used as bone replacement materials for the treatment of bone fractures and defects caused by osteoporosis. The aim of this study was to summarize current knowledge about the role of strontium in the treatment of osteoporosis, its effects (in various forms), and the ways in which it is administered.


Assuntos
Conservadores da Densidade Óssea/farmacologia , Osteoporose/tratamento farmacológico , Estrôncio/farmacologia , Animais , Conservadores da Densidade Óssea/uso terapêutico , Cálcio/metabolismo , Humanos , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Osteoporose/metabolismo , Estrôncio/uso terapêutico
12.
Int J Mol Sci ; 22(11)2021 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-34199520

RESUMO

Autosomal dominant polycystic kidney disease (ADPKD) is caused by loss of function of PKD1 (polycystin 1) or PKD2 (polycystin 2). The Ca2+-activated Cl- channel TMEM16A has a central role in ADPKD. Expression and function of TMEM16A is upregulated in ADPKD which causes enhanced intracellular Ca2+ signaling, cell proliferation, and ion secretion. We analyzed kidneys from Pkd1 knockout mice and found a more pronounced phenotype in males compared to females, despite similar levels of expression for renal tubular TMEM16A. Cell proliferation, which is known to be enhanced with loss of Pkd1-/-, was larger in male when compared to female Pkd1-/- cells. This was paralleled by higher basal intracellular Ca2+ concentrations in primary renal epithelial cells isolated from Pkd1-/- males. The results suggest enhanced intracellular Ca2+ levels contributing to augmented cell proliferation and cyst development in male kidneys. Enhanced resting Ca2+ also caused larger basal chloride currents in male primary cells, as detected in patch clamp recordings. Incubation of mouse primary cells, mCCDcl1 collecting duct cells or M1 collecting duct cells with dihydrotestosterone (DHT) enhanced basal Ca2+ levels and increased basal and ATP-stimulated TMEM16A chloride currents. Taken together, the more severe cystic phenotype in males is likely to be caused by enhanced cell proliferation, possibly due to enhanced basal and ATP-induced intracellular Ca2+ levels, leading to enhanced TMEM16A currents. Augmented Ca2+ signaling is possibly due to enhanced expression of Ca2+ transporting/regulating proteins.


Assuntos
Anoctamina-1/genética , Doenças Renais Policísticas/genética , Canais de Cátion TRPP/genética , Animais , Cálcio/metabolismo , Sinalização do Cálcio/genética , Proliferação de Células/genética , Cloretos/metabolismo , Di-Hidrotestosterona/farmacologia , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Rim/metabolismo , Rim/patologia , Masculino , Camundongos , Camundongos Knockout , Fenótipo , Doenças Renais Policísticas/metabolismo , Doenças Renais Policísticas/patologia , Caracteres Sexuais
13.
Int J Mol Sci ; 22(12)2021 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-34204292

RESUMO

Skin injury is quite common, and the wound healing is a complex process involving many types of cells, the extracellular matrix, and soluble mediators. Cell differentiation, migration, and proliferation are essential in restoring the integrity of the injured tissue. Despite the advances in science and technology, we have yet to find the ideal dressing that can support the healing of cutaneous wounds effectively, particularly for difficult-to-heal chronic wounds such as diabetic foot ulcers, bed sores, and venous ulcers. Hence, there is a need to identify and incorporate new ideas and methods to design a more effective dressing that not only can expedite wound healing but also can reduce scarring. Calcium has been identified to influence the wound healing process. This review explores the functions and roles of calcium in skin regeneration and reconstruction during would healing. Furthermore, this review also investigates the possibility of incorporating calcium into scaffolds and examines how it modulates cutaneous wound healing. In summary, the preliminary findings are promising. However, some challenges remain to be addressed before calcium can be used for cutaneous wound healing in clinical settings.


Assuntos
Cálcio/metabolismo , Cicatrização/fisiologia , Animais , Bandagens , Cálcio/farmacologia , Cálcio na Dieta/administração & dosagem , Fibroblastos/metabolismo , Humanos , Queratinócitos/metabolismo , Nanopartículas/química , Neovascularização Fisiológica , Regeneração , Pele/lesões , Pele/metabolismo , Nanomedicina Teranóstica , Engenharia Tecidual , Tecidos Suporte , Cicatrização/efeitos dos fármacos
14.
FASEB J ; 35(8): e21723, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34224609

RESUMO

Sperm acquire the ability to fertilize in a process called capacitation and undergo hyperactivation, a change in the motility pattern, which depends on Ca2+ transport by CatSper channels. CatSper is essential for fertilization and it is subjected to a complex regulation that is not fully understood. Here, we report that similar to CatSper, Cdc42 distribution in the principal piece is confined to four linear domains and this localization is disrupted in CatSper1-null sperm. Cdc42 inhibition impaired CatSper activity and other Ca2+ -dependent downstream events resulting in a severe compromise of the sperm fertilizing potential. We also demonstrate that Cdc42 is essential for CatSper function by modulating cAMP production by soluble adenylate cyclase (sAC), providing a new regulatory mechanism for the stimulation of CatSper by the cAMP-dependent pathway. These results reveal a broad mechanistic insight into the regulation of Ca2+ in mammalian sperm, a matter of critical importance in male infertility as well as in contraception.


Assuntos
Canais de Cálcio/metabolismo , Espermatozoides/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Animais , Cálcio/metabolismo , Canais de Cálcio/deficiência , Canais de Cálcio/genética , Sinalização do Cálcio , AMP Cíclico/metabolismo , Feminino , Fertilização In Vitro , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Transdução de Sinais , Capacitação Espermática/fisiologia , Motilidade Espermática/fisiologia , Cauda do Espermatozoide/metabolismo , Espermatozoides/efeitos dos fármacos , Espermatozoides/ultraestrutura , Proteína cdc42 de Ligação ao GTP/antagonistas & inibidores
15.
Nat Commun ; 12(1): 4293, 2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34257294

RESUMO

Mutations in the type 1 ryanodine receptor (RyR1), a Ca2+ release channel in skeletal muscle, hyperactivate the channel to cause malignant hyperthermia (MH) and are implicated in severe heat stroke. Dantrolene, the only approved drug for MH, has the disadvantages of having very poor water solubility and long plasma half-life. We show here that an oxolinic acid-derivative RyR1-selective inhibitor, 6,7-(methylenedioxy)-1-octyl-4-quinolone-3-carboxylic acid (Compound 1, Cpd1), effectively prevents and treats MH and heat stroke in several mouse models relevant to MH. Cpd1 reduces resting intracellular Ca2+, inhibits halothane- and isoflurane-induced Ca2+ release, suppresses caffeine-induced contracture in skeletal muscle, reduces sarcolemmal cation influx, and prevents or reverses the fulminant MH crisis induced by isoflurane anesthesia and rescues animals from heat stroke caused by environmental heat stress. Notably, Cpd1 has great advantages of better water solubility and rapid clearance in vivo over dantrolene. Cpd1 has the potential to be a promising candidate for effective treatment of patients carrying RyR1 mutations.


Assuntos
Bloqueadores dos Canais de Cálcio/uso terapêutico , Cálcio/metabolismo , Hipertermia Maligna/tratamento farmacológico , Hipertermia Maligna/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Animais , Halotano/farmacologia , Isoflurano/farmacologia , Camundongos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Mutação/genética
16.
Int J Mol Sci ; 22(12)2021 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-34204449

RESUMO

We recently found that, in human osteoblasts, Homer1 complexes to Calcium-sensing receptor (CaSR) and mediates AKT initiation via mechanistic target of rapamycin complex (mTOR) complex 2 (mTORC2) leading to beneficial effects in osteoblasts including ß-catenin stabilization and mTOR complex 1 (mTORC1) activation. Herein we further investigated the relationship between Homer1 and CaSR and demonstrate a link between the protein levels of CaSR and Homer1 in human osteoblasts in primary culture. Thus, when siRNA was used to suppress the CaSR, we observed upregulated Homer1 levels, and when siRNA was used to suppress Homer1 we observed downregulated CaSR protein levels using immunofluorescence staining of cultured osteoblasts as well as Western blot analyses of cell protein extracts. This finding was confirmed in vivo as the bone cells from osteoblast specific CaSR-/- mice showed increased Homer1 expression compared to wild-type (wt). CaSR and Homer1 protein were both expressed in osteocytes embedded in the long bones of wt mice, and immunofluorescent studies of these cells revealed that Homer1 protein sub-cellular localization was markedly altered in the osteocytes of CaSR-/- mice compared to wt. The study identifies additional roles for Homer1 in the control of the protein level and subcellular localization of CaSR in cells of the osteoblast lineage, in addition to its established role of mTORC2 activation downstream of the receptor.


Assuntos
Proteínas de Arcabouço Homer/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Osteoblastos/metabolismo , Receptores de Detecção de Cálcio/metabolismo , Animais , Cálcio/metabolismo , Linhagem da Célula , Sobrevivência Celular , Células Cultivadas , Feminino , Expressão Gênica , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Masculino , Camundongos , Camundongos Knockout , Fosforilação , Ligação Proteica , Transporte Proteico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Detecção de Cálcio/genética
17.
Int J Mol Sci ; 22(13)2021 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-34202925

RESUMO

Acetylcholine (ACh) is the classical neurotransmitter in the cholinergic nervous system. However, ACh is now known to regulate various immune cell functions. In fact, T cells, B cells, and macrophages all express components of the cholinergic system, including ACh, muscarinic, and nicotinic ACh receptors (mAChRs and nAChRs), choline acetyltransferase, acetylcholinesterase, and choline transporters. In this review, we will discuss the actions of ACh in the immune system. We will first briefly describe the mechanisms by which ACh is stored in and released from immune cells. We will then address Ca2+ signaling pathways activated via mAChRs and nAChRs on T cells and B cells, highlighting the importance of ACh for the function of T cells, B cells, and macrophages, as well as its impact on innate and acquired (cellular and humoral) immunity. Lastly, we will discuss the effects of two peptide ligands, secreted lymphocyte antigen-6/urokinase-type plasminogen activator receptor-related peptide-1 (SLURP-1) and hippocampal cholinergic neurostimulating peptide (HCNP), on cholinergic activity in T cells. Overall, we stress the fact that ACh does not function only as a neurotransmitter; it impacts immunity by exerting diverse effects on immune cells via mAChRs and nAChRs.


Assuntos
Imunomodulação , Receptores Muscarínicos/metabolismo , Receptores Nicotínicos/metabolismo , Acetilcolina/metabolismo , Animais , Cálcio/metabolismo , Sinalização do Cálcio , Regulação da Expressão Gênica , Humanos , Sistema Imunitário/imunologia , Sistema Imunitário/metabolismo , Imunidade , Linfócitos/imunologia , Linfócitos/metabolismo , Especificidade de Órgãos , Peptídeos/metabolismo , Peptídeos/farmacologia , Receptores Muscarínicos/genética , Receptores Nicotínicos/genética , Transdução de Sinais
18.
Int J Mol Sci ; 22(13)2021 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-34209674

RESUMO

Pre-mRNA processing factor 4B (PRP4) has previously been shown to induce epithelial-mesenchymal transition (EMT) and drug resistance in cancer cell lines. As melanin plays an important photoprotective role in the risk of sun-induced skin cancers, we have investigated whether PRP4 can induce drug resistance and regulate melanin biosynthesis in a murine melanoma (B16F10) cell line. Cells were incubated with a crucial melanogenesis stimulator, alpha-melanocyte-stimulating hormone, followed by transfection with PRP4. This resulted in the inhibition of the production of melanin via the downregulation of adenylyl cyclase-cyclic adenosine 3',5'-monophosphate (AC)-(cAMP)-tyrosinase synthesis signaling pathway. Inhibition of melanin production by PRP4 leads to the promotion of carcinogenesis and induced drug resistance in B16F10 cells. Additionally, PRP4 overexpression upregulated the expression of ß-arrestin 1 and desensitized the extracellular calcium-sensing receptor (CaSR), which in turn, inhibited the influx of extracellular Ca2+ ions. The decreased influx of Ca2+ was confirmed by a decreased expression level of calmodulin. We have demonstrated that transient receptor potential cation channel subfamily C member 1 was involved in the influx of CaSR-induced Ca2+ via a decreasing level of its expression. Furthermore, PRP4 overexpression downregulated the expression of AC, decreased the synthesis of cAMP, and modulated the actin cytoskeleton by inhibiting the expression of Ras homolog family member A (RhoA). Our investigation suggests that PRP4 inhibits the production of melanin in B16F10 cells, blocks the influx of Ca2+ through desensitization of CaSR, and modulates the actin cytoskeleton through downregulating the AC-cAMP pathway; taken together, these observations collectively lead to the promotion of skin carcinogenesis.


Assuntos
Citoesqueleto de Actina/metabolismo , Cálcio/metabolismo , Melaninas/biossíntese , Ribonucleoproteína Nuclear Pequena U4-U6/metabolismo , Neoplasias Cutâneas/etiologia , Neoplasias Cutâneas/metabolismo , Animais , Antineoplásicos , Calmodulina/genética , Calmodulina/metabolismo , Proteínas de Transporte , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Espaço Extracelular/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Espaço Intracelular/metabolismo , Melanoma Experimental , Camundongos , Ligação Proteica , Ribonucleoproteína Nuclear Pequena U4-U6/genética , Transdução de Sinais/efeitos dos fármacos , Neoplasias Cutâneas/patologia
19.
Int J Mol Sci ; 22(13)2021 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-34281258

RESUMO

Infection by Proteus mirabilis causes urinary stones and catheter incrustation due to ammonia formed by urease (PMU), one of its virulence factors. Non-enzymatic properties, such as pro-inflammatory and neurotoxic activities, were previously reported for distinct ureases, including that of the gastric pathogen Helicobacter pylori. Here, PMU was assayed on isolated cells to evaluate its non-enzymatic properties. Purified PMU (nanomolar range) was tested in human (platelets, HEK293 and SH-SY5Y) cells, and in murine microglia (BV-2). PMU promoted platelet aggregation. It did not affect cellular viability and no ammonia was detected in the cultures' supernatants. PMU-treated HEK293 cells acquired a pro-inflammatory phenotype, producing reactive oxygen species (ROS) and cytokines IL-1ß and TNF-α. SH-SY5Y cells stimulated with PMU showed high levels of intracellular Ca2+ and ROS production, but unlike BV-2 cells, SH-SY5Y did not synthesize TNF-α and IL-1ß. Texas Red-labeled PMU was found in the cytoplasm and in the nucleus of all cell types. Bioinformatic analysis revealed two bipartite nuclear localization sequences in PMU. We have shown that PMU, besides urinary stone formation, can potentially contribute in other ways to pathogenesis. Our data suggest that PMU triggers pro-inflammatory effects and may affect cells beyond the renal system, indicating a possible role in extra-urinary diseases.


Assuntos
Proteus mirabilis/enzimologia , Proteus mirabilis/patogenicidade , Urease/metabolismo , Urease/toxicidade , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Linhagem Celular , Núcleo Celular/metabolismo , Células HEK293 , Humanos , Técnicas In Vitro , Camundongos , Microglia/efeitos dos fármacos , Microglia/metabolismo , Microglia/microbiologia , Modelos Moleculares , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/microbiologia , Neurotoxinas/química , Neurotoxinas/metabolismo , Neurotoxinas/toxicidade , Sinais de Localização Nuclear , Agregação Plaquetária/efeitos dos fármacos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/toxicidade , Urease/química , Virulência/fisiologia
20.
Int J Mol Sci ; 22(10)2021 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-34063554

RESUMO

Acute lung injury (ALI) afflicts approximately 200,000 patients annually and has a 40% mortality rate. The COVID-19 pandemic has massively increased the rate of ALI incidence. The pathogenesis of ALI involves tissue damage from invading microbes and, in severe cases, the overexpression of inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß). This study aimed to develop a therapy to normalize the excess production of inflammatory cytokines and promote tissue repair in the lipopolysaccharide (LPS)-induced ALI. Based on our previous studies, we tested the insulin-like growth factor I (IGF-I) and BTP-2 therapies. IGF-I was selected, because we and others have shown that elevated inflammatory cytokines suppress the expression of growth hormone receptors in the liver, leading to a decrease in the circulating IGF-I. IGF-I is a growth factor that increases vascular protection, enhances tissue repair, and decreases pro-inflammatory cytokines. It is also required to produce anti-inflammatory 1,25-dihydroxyvitamin D. BTP-2, an inhibitor of cytosolic calcium, was used to suppress the LPS-induced increase in cytosolic calcium, which otherwise leads to an increase in proinflammatory cytokines. We showed that LPS increased the expression of the primary inflammatory mediators such as toll like receptor-4 (TLR-4), IL-1ß, interleukin-17 (IL-17), TNF-α, and interferon-γ (IFN-γ), which were normalized by the IGF-I + BTP-2 dual therapy in the lungs, along with improved vascular gene expression markers. The histologic lung injury score was markedly elevated by LPS and reduced to normal by the combination therapy. In conclusion, the LPS-induced increases in inflammatory cytokines, vascular injuries, and lung injuries were all improved by IGF-I + BTP-2 combination therapy.


Assuntos
Lesão Pulmonar Aguda/tratamento farmacológico , Lesão Pulmonar Aguda/metabolismo , Anilidas/farmacologia , Citocinas/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Fator de Crescimento Insulin-Like I/farmacologia , Tiadiazóis/farmacologia , Lesão Pulmonar Aguda/patologia , Lesão Pulmonar Aguda/virologia , Anilidas/uso terapêutico , Animais , COVID-19/complicações , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Citocinas/genética , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica/genética , Imuno-Histoquímica , Fator de Crescimento Insulin-Like I/metabolismo , Fator de Crescimento Insulin-Like I/uso terapêutico , Interferon gama/genética , Interferon gama/metabolismo , Interleucina-17/genética , Interleucina-17/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Lipopolissacarídeos/toxicidade , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Tiadiazóis/uso terapêutico , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
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