Myristoylated Alanine-rich C-kinase Substrate mRNA Research Articles

Mir-21 Regulation of MARCKS Protein and Mucin Secretion in Airway Epithelial Cells

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.

Expression of protein kinase-C substrate mRNA in the motor cortex of adult and infant macaque monkeys

To understand the molecular and cellular bases of plasticity in the primate motor cortex, we investigated the expression of three protein kinase-C (PKC) substrates: GAP-43, myristoylated alanine-rich C-kinase substrate (MARCKS), and neurogranin, which are key molecules regulating synaptic plasticity. Prominent signals for the three mRNAs were primarily observed in pyramidal cells. Large pyramidal cells in layer V, from which the descending motor tract originates, contained weaker hybridization signals for GAP-43 and neurogranin mRNAs than did the smaller pyramidal cells. We also performed double-label in situ hybridization showing that GAP-43 and neurogranin mRNAs were expressed in a subset of MARCKS-positive neurons. Quantitative analysis showed that the expression was different between the layers: layer VI contained the strongest and layer II the weakest signals for all three mRNAs. The expression levels of GAP-43 and MARCKS mRNA in layer V were higher than in layer III, while the expression level of neurogranin mRNA in layer V was almost the same as in layer III. Developmental analysis from the newborn to adult indicated that the expression levels of the three mRNAs were higher in the infant motor cortex than in the adult. The expression of both GAP-43 and neurogranin mRNAs transiently increased over several months postnatally. The present study showed that the expression of the three PKC substrates was specific to cell types, cortical layers, and postnatal developmental stage. The specific expression may reflect functional specialization for plasticity in the motor cortex of both infants and adults.

Northern blot and in situ hybridization analyses for the development of myristoylated alanine-rich c-kinase substrate mRNA in the monkey cerebral cortex

Myristoylated alanine-rich C-kinase substrate (MARCKS) is a major neuron-specific substrate for protein kinase C, and is involved in both neurite outgrowth and synaptic plasticity. Using both Northern blot and in situ hybridization techniques, we investigated whether the expression of MARCKS mRNA in the monkey cerebral neocortex and hippocampus changed during the developmental period. In each of four neocortical areas examined, i.e. the prefrontal area (area FD of [Illinois Monographs in the Medical Sciences (1947) 1]), the temporal association area (TE), the primary somatosensory area (PB), and the primary visual area (OC), the Northern blot analysis showed that the amount of MARCKS mRNA was high during the fetal and early postnatal periods, and decreased sharply between postnatal day 70 and postnatal month 6. The in situ hybridization experiments showed that the expression of MARCKS mRNA was decreased in every layer of neocortical areas at postnatal month 6 or later. In the primary sensory areas (areas PB and OC), the degree of decrease was higher in the supragranular layers (layers II and III) than in the infragranular layers (layers V and VI). In the hippocampus, the developmental change in the amount of MARCKS mRNA was small, but the in situ hybridization revealed a prominent decrease in Ammon's horn in monkeys on postnatal month 8 and later. These findings indicate that region-specific expression of MARCKS mRNA is established around postnatal month 6. We suggest that the extensive expression of MARCKS mRNA is one of the molecular bases of high plasticity in the infant cerebral cortex.

Expression of MARCKS mRNA in lateral geniculate nucleus and visual cortex of normal and monocularly deprived macaque monkeys.

We performed a nonradioactive in situ hybridization histochemistry (ISH) study of the lateral geniculate nucleus (LGN) and the primary visual area (area 17) of the macaque monkey to investigate mRNA expression of the myristoylated alanine-rich C-kinase substrate (MARCKS), a major protein kinase C (PKC) substrate. In the LGN, intense hybridization signals were observed in both magnocellular neurons (layers 1 and 2) and parvocellular neurons (layers 3 to 6). Double labeling using ISH and immunofluorescence revealed that MARCKS mRNA was coexpressed with the alpha-subunit of type II calcium/calmodulin-dependent protein kinase, indicating that MARCKS mRNA is also expressed in koniocellular neurons in the LGN. GABA-immunoreactive neurons in the LGN did not contain MARCKS mRNA, indicating that MARCKS mRNA is not expressed in inhibitory interneurons. The signals were generally weak in area 17, and intense signals were restricted to large neurons in layers IVB, V, and VI. GABA-immunoreactive neurons in layers II-VI of area 17 did not contain MARCKS mRNA. Double-label ISH revealed that MARCKS mRNA was coexpressed with mRNA of GAP-43, another PKC substrate, in neurons of both the LGN and area 17. To determine whether the expression of MARCKS mRNA is regulated by retinal activity, we performed ISH in the LGN and area 17 of monkeys deprived of monocular visual input by tetrodotoxin. After monocular deprivation for 5 to 30 days, MARCKS mRNA was down-regulated in the LGN, but not in area 17. These results suggest that MARCKS mediates the activity-dependent changes in the excitatory relay neurons in the LGN.

Myristoylated alanine-rich C kinase substrate protein and mRNA in bovine corpus luteum during the estrous cycle.

The bovine corpus luteum contains a myristoylated alanine-rich C kinase substrate (MARCKS) protein known to crosslink actin filaments in the cytoskeletal cortex associated with the plasma membrane. We conducted experiments to determine whether concentrations of MARCKS mRNA and protein in the bovine corpus luteum varied during the estrous cycle. Using Northern blots probed with a MARCKS cDNA, we found that luteal concentrations of MARCKS mRNA were greatest on d 4, 8, and 12 and markedly reduced on d 16 of the cycle (p < 0.08). Similarly, Western blot analysis of luteal proteins revealed that concentrations of MARCKS protein were greatest on d 8 and least on d 16 of the cycle (p < 0.01). Exposure of slices from a d 8 corpus luteum to prostaglandin F2alpha (PGF2alpha) during a 10-min incubation in the presence of [32P]-ortho-phosphate resulted in enhanced phosphorylation of MARCKS in membrane and cytosolic fractions compared to that of controls. We therefore concluded that expression of the luteal MARCKS protein gene may be regulated and that PGF2alpha-induced phosphorylation of this protein is attributable to activation of protein kinase C.

MARCKS functions as a novel growth suppressor in cells of melanocyte origin.

Protein kinase C (PKC) plays a pivotal role in modulating the growth of melanocytic cells in culture. We have shown previously that a major physiological substrate of PKC, the 80 kDa myristoylated alanine-rich C-Kinase substrate (MARCKS), can be phosphorylated in quiescent, non-tumorigenic melanocytes exposed transiently to a biologically active phorbol ester, but cannot be phosphorylated in phorbol ester-treated, syngeneic malignant melanoma cells. Despite its ubiquitous distribution, the function of MARCKS in cell growth and transformation remains to be demonstrated clearly. We report here that MARCKS mRNA and protein levels are down-regulated significantly in the spontaneously derived murine B16 melanoma cell line compound with syngeneic normal Mel-ab melanocytes. In contrast, the tumourigenic v-Ha-ras-transformed melanocytic line, LTR Ras 2, showed a high basal level of MARCKS phosphorylation which was not enhanced by treatment of cells with phorbol ester. Furthermore, protein levels of MARCKS in LTR Ras 2 cells were similar to those expressed in Mel-ab melanocytes. However, in four out of six murine tumour cell lines investigated, levels of MARCKS protein were barely detectable. Transfection of B16 cells with a plasmid containing the MARCKS cDNA in the sense orientation produced two neomycin-resistant clones displaying reduced proliferative capacity and decreased anchorage-independent growth compared with control cells. In contrast, transfection with the antisense MARCKS construct produced many colonies which displayed enhanced growth and transforming potential compared with control cells. Thus, MARCKS appears to act as a novel growth suppressor in the spontaneous transformation of cells of melanocyte origin and may play a more general role in the tumour progression of other carcinoma.