Hypersecretion of mucus characterizes many inflammatory airway diseases, including asthma, chronic bronchitis, and cystic fibrosis. Excess mucus causes airway obstruction, reduces pulmonary function, and can lead to i...Hypersecretion of mucus characterizes many inflammatory airway diseases, including asthma, chronic bronchitis, and cystic fibrosis. Excess mucus causes airway obstruction, reduces pulmonary function, and can lead to increased morbidity and mortality. MicroRNAs are small non-coding pieces of RNA which regulate other genes by binding to a complementary sequence in the target mRNA. The microRNA miR-21 is upregulated in many inflammatory conditions and, interestingly, miR-21 has been shown to target the mRNA of Myristoylated Alanine-Rich C Kinase Substrate (MARCKS), a protein that is an important regulator of airway mucin (the solid component of mucus) secretion. In these studies, we determined that exposure of primary, well-differentiated, normal human bronchial epithelial (NHBE) cells to the pro-inflammatory stimulus lipopolysaccharide (LPS) increased expression of both miR-21 and MARCKS in a time-dependent manner. To investigate whether miR-21 regulation of MARCKS played a role in mucin secretion, two separate airway epithelial cell lines, HBE1 (papilloma virus transformed) and NCI-H292 (mucodepidermoid derived) were utilized, since manipulation of miR-21 is performed via transfection of commercially-available miR-21 inhibitors and mimics/activators. Treatment of HBE1 cells with LPS caused concentration-dependent increases in expression of both miR-21 and MARCKS mRNA and protein. The miR-21 inhibitor effectively reduced levels of miR-21 in the cells, coincident with an increase in MARCKS mRNA expression over time as well as enhanced mucin secretion, while the miR-21 mimic/activator increased levels of miR-21, which coincided with a decrease in expression of MARCKS and a decrease in mucin secretion. These results suggest that miR-21 is increased in airway epithelial cells following exposure to LPS, and that miR-21 downregulates expression of MARCKS, which may decrease mucin secretion by the cells. Thus, miR-21 may act as a negative feedback regulator of mucin secretion in airway epithelial cells, and may do so, at least in part, by downregulating expression of MARCKS.展开更多
Cell proliferation, differentiation, and the elimination of unnecessary cells by apoptosis occur in the development of the nervous system. It is reported that brain dysplasia appears as the results of myristoylated al...Cell proliferation, differentiation, and the elimination of unnecessary cells by apoptosis occur in the development of the nervous system. It is reported that brain dysplasia appears as the results of myristoylated alanine-rich C kinase substrate (MARCKS) knockout or the mutant mouse. We therefore expect that MARCKS participates in the development of the nervous system. However, the mechanism underlying such participation has not been identified. In this study, we observed the effects of the overexpression of MARCKS or unphosphorylatable MARCKS on cell proliferation and TNF-α-induced apoptosis in neuroblastoma SH-SY5Y cells. Furthermore, we restrained MARCKS expression by the RNAi method. In the results, MARCKS-overexpressing cells and not unphosphorylatable MARCKS-overexpressing cells showed increased cell proliferation rates. On the other hand, the RNAi decreased the proliferation of MARCKS-knocked down SH-SY5Y cells. These results indicated that MARCKS-overexpressing cells were more sensitive to TNF-α than normal SH-SY5Y cells. Moreover, in MARCKS-overexpressing cells TNF-α-induced apoptosis was inhibited by caspase-6 and -7 inhibitors but not by caspase-3 inhibitor. These results suggested that MARCKS participated in TNF-α-induced apoptosis in a caspase-6 and/or -7-dependent manner.展开更多
Inflammation, which is induced after infection of bacteria and tissue injury, is one of the important early stages of wound healing. Bradykinin is increased during acute and chronic inflammation. We previously reporte...Inflammation, which is induced after infection of bacteria and tissue injury, is one of the important early stages of wound healing. Bradykinin is increased during acute and chronic inflammation. We previously reported that bradykinin stimulation induces dephosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS) after phosphorylation by ROCK leading neurite outgrowth in neuroblastoma SH-SY5Y cells. In this report we showed that knock-down of MARCKS by RNAi reduced cell migration. Wild-type MARCKS-overexpressed SH-SY5Y cells migrated faster than the control cells. Unphosphorylatable MARCKS-overexpressed cells notably migrated fast. Moreover, chronic MARCKS dephosphorylation by a ROCK inhibitor HA-1077 promoted the cell migration, on the other hand a PKC inhibitor Ro-31-8220 did not. After wounding, MARCKS was transiently phosphorylated and dephospho-rylated by 20 min. Immunocytochemistry showed that the dephosphorylated MARCKS was localized at neurite tips. These findings suggest that MARCKS dephosphorylation is important in wound-induced migration of SH-SY5Y cells. It indicates the possibility that MARCKS is associated with wound repair after inflammation.展开更多
文摘Hypersecretion of mucus characterizes many inflammatory airway diseases, including asthma, chronic bronchitis, and cystic fibrosis. Excess mucus causes airway obstruction, reduces pulmonary function, and can lead to increased morbidity and mortality. MicroRNAs are small non-coding pieces of RNA which regulate other genes by binding to a complementary sequence in the target mRNA. The microRNA miR-21 is upregulated in many inflammatory conditions and, interestingly, miR-21 has been shown to target the mRNA of Myristoylated Alanine-Rich C Kinase Substrate (MARCKS), a protein that is an important regulator of airway mucin (the solid component of mucus) secretion. In these studies, we determined that exposure of primary, well-differentiated, normal human bronchial epithelial (NHBE) cells to the pro-inflammatory stimulus lipopolysaccharide (LPS) increased expression of both miR-21 and MARCKS in a time-dependent manner. To investigate whether miR-21 regulation of MARCKS played a role in mucin secretion, two separate airway epithelial cell lines, HBE1 (papilloma virus transformed) and NCI-H292 (mucodepidermoid derived) were utilized, since manipulation of miR-21 is performed via transfection of commercially-available miR-21 inhibitors and mimics/activators. Treatment of HBE1 cells with LPS caused concentration-dependent increases in expression of both miR-21 and MARCKS mRNA and protein. The miR-21 inhibitor effectively reduced levels of miR-21 in the cells, coincident with an increase in MARCKS mRNA expression over time as well as enhanced mucin secretion, while the miR-21 mimic/activator increased levels of miR-21, which coincided with a decrease in expression of MARCKS and a decrease in mucin secretion. These results suggest that miR-21 is increased in airway epithelial cells following exposure to LPS, and that miR-21 downregulates expression of MARCKS, which may decrease mucin secretion by the cells. Thus, miR-21 may act as a negative feedback regulator of mucin secretion in airway epithelial cells, and may do so, at least in part, by downregulating expression of MARCKS.
文摘Cell proliferation, differentiation, and the elimination of unnecessary cells by apoptosis occur in the development of the nervous system. It is reported that brain dysplasia appears as the results of myristoylated alanine-rich C kinase substrate (MARCKS) knockout or the mutant mouse. We therefore expect that MARCKS participates in the development of the nervous system. However, the mechanism underlying such participation has not been identified. In this study, we observed the effects of the overexpression of MARCKS or unphosphorylatable MARCKS on cell proliferation and TNF-α-induced apoptosis in neuroblastoma SH-SY5Y cells. Furthermore, we restrained MARCKS expression by the RNAi method. In the results, MARCKS-overexpressing cells and not unphosphorylatable MARCKS-overexpressing cells showed increased cell proliferation rates. On the other hand, the RNAi decreased the proliferation of MARCKS-knocked down SH-SY5Y cells. These results indicated that MARCKS-overexpressing cells were more sensitive to TNF-α than normal SH-SY5Y cells. Moreover, in MARCKS-overexpressing cells TNF-α-induced apoptosis was inhibited by caspase-6 and -7 inhibitors but not by caspase-3 inhibitor. These results suggested that MARCKS participated in TNF-α-induced apoptosis in a caspase-6 and/or -7-dependent manner.
文摘Inflammation, which is induced after infection of bacteria and tissue injury, is one of the important early stages of wound healing. Bradykinin is increased during acute and chronic inflammation. We previously reported that bradykinin stimulation induces dephosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS) after phosphorylation by ROCK leading neurite outgrowth in neuroblastoma SH-SY5Y cells. In this report we showed that knock-down of MARCKS by RNAi reduced cell migration. Wild-type MARCKS-overexpressed SH-SY5Y cells migrated faster than the control cells. Unphosphorylatable MARCKS-overexpressed cells notably migrated fast. Moreover, chronic MARCKS dephosphorylation by a ROCK inhibitor HA-1077 promoted the cell migration, on the other hand a PKC inhibitor Ro-31-8220 did not. After wounding, MARCKS was transiently phosphorylated and dephospho-rylated by 20 min. Immunocytochemistry showed that the dephosphorylated MARCKS was localized at neurite tips. These findings suggest that MARCKS dephosphorylation is important in wound-induced migration of SH-SY5Y cells. It indicates the possibility that MARCKS is associated with wound repair after inflammation.
