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Downstream Mechanism (downstream + mechanism)
Selected AbstractsProtein kinase C , phosphorylates keratin 8 at Ser8 and Ser23 in GH4C1 cells stimulated by thyrotropin-releasing hormoneFEBS JOURNAL, Issue 13 2007Yoshiko Akita Protein kinase C , (PKC,) is activated by thyrotropin-releasing hormone (TRH), a regulator of pituitary function in rat pituitary GH4C1 cells. We analyzed the downstream mechanism after PKC, activation. Exposure of GH4C1 cells to TRH or a phorbol ester increased the phosphorylation of three p52 proteins (p52a, p52b and p52c) and decreased the phosphorylation of destrin and cofilin. GF109203X, an inhibitor of protein kinases including PKC, inhibited phosphorylation of the p52 proteins by TRH stimulation. Peptide mapping, amino-acid sequencing, and immunochemical studies indicated that p52a, p52b, and p52c are the differentially phosphorylated isoforms of keratin 8 (K8), an intermediate filament protein. The unphosphorylated K8 (p52n) localized exclusively to the cytoskeleton, whereas the phosphorylated forms (especially p52c), which are increased in TRH-stimulated cells, localized mainly to the cytosol. K8 phosphorylation was enhanced in PKC,-overexpressing clones, and purified recombinant PKC, directly phosphorylated K8 with a profile similar to that observed in TRH-stimulated cells. PKC, and K8 colocalized near the nucleus under basal conditions and were concentrated in the cell periphery and cell,cell contact area after TRH stimulation. MS analyses of phospho-K8 and K8-synthesized peptide (amino acids 1,53) showed that PKC, phosphorylates Ser8 and Ser23 of K8. Phosphorylation of these sites is enhanced in TRH-stimulated cells and PKC,-overexpressing cells, as assessed by immunoblotting using antibodies to phospho-K8. These results suggest that K8 is a physiological substrate for PKC,, and the phosphorylation at Ser8 and Ser23 transduces, at least in part, TRH,PKC, signaling in pituitary cells. [source] Expression of Acid-Sensing Ion Channel 3 (ASIC3) in Nucleus Pulposus Cells of the Intervertebral Disc Is Regulated by p75NTR and ERK Signaling,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 12 2007Yoshiyasu Uchiyama Abstract Although a recent study has shown that skeletal tissues express ASICs, their function is unknown. We show that intervertebral disc cells express ASIC3; moreover, expression is uniquely regulated and needed for survival in a low pH and hypoeromsotic medium. These findings suggest that ASIC3 may adapt disc cells to their hydrodynamically stressed microenvironment. Introduction: The nucleus pulposus is an avascular, hydrated tissue that permits the intervertebral disc to resist compressive loads to the spine. Because the tissue is hyperosmotic and avascular, the pH of the nucleus pulposus is low. To determine the mechanisms by which the disc cells accommodate to the low pH and hypertonicity, the expression and regulation of the acid sensing ion channel (ASIC)3 was examined. Materials and Methods: Expression of ASICs in cells of the intervertebral disc was analyzed. To study its regulation, we cloned the 2.8-kb rat ASIC3 promoter and performed luciferase reporter assays. The effect of pharmacological inhibition of ASICs on disc cell survival was studied by measuring MTT and caspase-3 activities. Results: ASIC3 was expressed in discal tissues and cultured disc cells in vitro. Because studies of neuronal cells have shown that ASIC3 expression and promoter activity is induced by nerve growth factor (NGF), we examined the effect of NGF on nucleus pulposus cells. Surprisingly, ASIC3 promoter activity did not increase after NGF treatment. The absence of induction was linked to nonexpression of tropomyosin-related kinase A (TrkA), a high-affinity NGF receptor, although a modest expression of p75NTR was seen. When treated with p75NTR antibody or transfected with dominant negative-p75NTR plasmid, there was significant suppression of ASIC3 basal promoter activity. To further explore the downstream mechanism of control of ASIC3 basal promoter activity, we blocked p75NTR and measured phospho extracellular matrix regulated kinase (pERK) levels. We found that DN-p75NTR suppressed NGF mediated transient ERK activation. Moreover, inhibition of ERK activity by dominant negative-mitogen activated protein kinase kinase (DN-MEK) resulted in a dose-dependent suppression of ASIC3 basal promoter activity, whereas overexpression of constitutively active MEK1 caused an increase in ASIC3 promoter activity. Finally, to gain insight in the functional importance of ASIC3, we suppressed ASIC activity in nucleus pulposus cells. Noteworthy, under both hyperosmotic and acidic conditions, ASIC3 served to promote cell survival and lower the activity of the pro-apoptosis protein, caspase-3. Conclusions: Results of this study indicate that NGF serves to maintain the basal expression of ASIC3 through p75NTR and ERK signaling in discal cells. We suggest that ASIC3 is needed for adaptation of the nucleus pulposus and annulus fibrosus cells to the acidic and hyperosmotic microenvironment of the intervertebral disc. [source] The role of activins during decidualisation of human endometriumAUSTRALIAN AND NEW ZEALAND JOURNAL OF OBSTETRICS AND GYNAECOLOGY, Issue 3 2006Rebecca L. JONES Decidualisation of the endometrial stroma is critical to create a specialised environment for embryo implantation and trophoblast invasion; however, the mechanisms involved are poorly understood. We have established that activin A is an important regulator of decidualisation of endometrial stromal cells in vitro. Here we describe studies that verify the physiological significance of these findings. We demonstrate that high concentrations of activin A are produced by decidualising cells in excess of the antagonists, inhibin and follistatin, thus confirming its bioavailability within the decidual environment. Furthermore, we demonstrate that all components of the activin signalling pathway (activin receptors and Smads) are expressed in decidualised cells, and identify a downstream mechanism for activin in the endometrium, through the regulation of matrix metalloproteinases (MMPs). This new knowledge is important for understanding the roles for activins and inhibins in regulating fertility. [source] Glial aromatization increases the expression of bone morphogenetic protein-2 in the injured zebra finch brainJOURNAL OF NEUROCHEMISTRY, Issue 1 2008Bradley J. Walters Abstract In songbirds, brain injury upregulates glial aromatase. The resulting local estrogen synthesis mitigates apoptosis and enhances cytogenesis by poorly understood mechanisms. Bone morphogenetic proteins (BMPs), long studied for their role in neural development, are also neuroprotective and cytogenic in the adult brain. BMPs remain uncharacterized in songbirds, as do the mechanisms regulating their post-injury expression. We first established the expression of BMPs 2, 4, 6, and 7 in the adult zebra finch brain using RT-PCR. Next, we determined the effect of neural insult on BMP expression, by comparing BMP transcripts between injured and uninjured telencephalic hemispheres using semi-quantitative PCR. The expression of BMPs 2 and 4, but not 6 and 7, increased 24 h post-injury. To determine the influence of aromatase on BMP expression, we compared BMP expression following delivery of the aromatase inhibitor Fadrozole or vehicle into contralateral hemispheres. Fadrozole decreased BMP2, but not BMP4, expression, suggesting that aromatization may induce BMP2 expression following injury. Since BMPs are gliogenic and neurotrophic, future studies will test if the neuroprotective and cytogenic effects of aromatase upregulation are mediated by BMP2. Songbirds may be excellent models towards understanding the role of local estrogen synthesis and its downstream mechanisms on neuroprotection and repair. [source] Mechanisms of acetylcholine-mediated vasodilatation in young and aged human skinTHE JOURNAL OF PHYSIOLOGY, Issue 3 2005Lacy A. Holowatz Thermoregulatory cutaneous vasodilatation (VD) is attenuated in aged skin. While acetylcholine (ACh) plays a role in thermally mediated VD, the precise mechanisms through which ACh-mediated VD acts and whether those downstream mechanisms change with ageing are unclear. We tested the hypotheses that both nitric oxide (NO)- and prostanoid-mediated pathways contribute to exogenous ACh-mediated VD, and that both are attenuated with advanced age. Twelve young (Y: 23 ± 1 years) and 10 older (O: 69 ± 1 years) subjects underwent infusions of 137.5 ,m ACh at four intradermal microdialysis sites: control (C, Ringer solution), NO synthase inhibited (NOS-I, 10 mml -NAME), cyclooxygenase inhibited (COX-I, 10 mm ketorolac) and NOS-I + COX-I. Red blood cell flux was monitored using laser-Doppler flowmetry, and cutaneous vascular conductance (CVC) was calculated (laser-Doppler flux/mean arterial pressure) and normalized to maximal CVC (%CVCmax) (28 mm sodium nitroprusside + local heating to 43°C). Baseline %CVCmax was increased in the O at COX-I sites (COX-I 16 ± 1, NOS-I + COX-I 16 ± 2 versus C 10 ± 1%CVCmax; P < 0.001) but not in the young, suggesting an age-related shift toward COX vasoconstrictors contributing to basal cutaneous vasomotor tone. There was no difference in peak %CVCmax during ACh infusion between age groups, and the response was unchanged by NOS-I (O: NOS-I 35 ± 5 versus C 38 ± 5%CVCmax; P= 0.84) (Y: NOS-I 41 ± 4 versus C 39 ± 4%CVCmax; P= 0.67). COX-I and NOS-I + COX-I attenuated the peak CVC response to ACh in both groups (COX-I O: 29 ± 3, Y: 22 ± 2%CVCmaxversus C; P < 0.001 both groups; NOS-I + COX-I O: 32 ± 3 versus Y: 29 ± 2%CVCmax; versus C; P < 0.001 both groups). ACh mediates cutaneous VD through prostanoid and non-NO-, non-prostanoid-dependent pathways. Further, older subjects have a diminished prostanoid contribution to ACh-mediated VD. [source] | |||||||||||||