Browsing by Subject "Mitogen-Activated Protein Kinases"
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Item NF-kB Mediates Cartilage Degradation induced by Trauma Injury and IL-1(2013-01-22) Carter, Kristen; Kashyap, Meghana; Sauer, Brent C.; Chen, Christopher T.BACKGROUND: IL-1 is one of the major pro-inflammatory cytokines responsible for cartilage degradation. Several studies have shown that IL-1 mediates the upregulation of tissue degradation through the NF-κB and Mitogen-activated protein kinase (MAPK, p38) pathways, but its role in cartilage degradation after blunt trauma injury is not clear. The objective of this study was to determine the roles of NF-κB and p38 in IL-1- induced cell death, proteoglycan (PG) degradation, nitric oxide (NO) production, and related gene upregulation in cartilage after blunt injury. METHODS: Full-thickness cartilage plugs were obtained from mature bovine knees (>18 mo) and pre-cultured in DMEM. The signaling pathways (p38 and NF-kB) were inhibited by pretreatment with 10μM SB202190 for p38 (p38i) and 50μM BAY117085 for NF-κB (IκBi) for 1 hour. Samples in Injury and Injury+IL-1 groups received impact injury with impact energy of 15J/cm2. IL-1 and Injury+IL-1 groups were treated with 1 ng/ml IL-1. Cell viability was assessed using fluorescein diacetate and propidium iodine. The mRNA from cartilage was isolated using Trizol and RNeasy Mini kit (Qiagen), reverse transcribed, and analyzed using qPCR to determine pro-inflammatory cytokine and tissue remodeling genes (IL- 6, MMP-3, TIMP-3). All gene expression was normalized to GAPDH. The medium was analyzed for proteoglycan (PG) release/loss and nitric oxide (NO) production using dimethylmethylene blue (DMMB) and Greiss assays, respectively. RESULTS: Increased cell death was found in the Injury and Injuy+IL-1 groups. Increase of PG loss was found in IL-1, Injury and Injury+IL-1 treated groups (37%, 104% and 126%, respectively). Significant decreases (69-73%) of PG loss were found in all IκBi treated groups, while little or no changes were found in the p38 groups. Results from the qPCR analysis supported the findings. IκBi treatment reduced MMP-3 upregulation induced by IL-1 and Injury, while there was minimal change with the p38 inhibitor. Similarly, NO production was also decreased in the IκBi treated groups. DISCUSSION and CONCLUSION: Our study suggests that the NF-κB signaling pathway plays a greater role than p38 in IL-1 mediated PG loss and NO production in cartilage after trauma injury. Future studies are needed to determine the time-course response and specific NF-κB mediators for downstream regulation, as well as the effects in long-term therapeutic treatment to ameliorate the progress of post traumatic osteoarthritis.Item The Regulation of Autophagy and Its Role in Mitotic Exit(2014-07-14) An, Zhenyi; Tu, Benjamin; Cobb, Melanie H.; White, Michael A.Autophagy is an evolutionarily conserved pathway in which cells enclose cytoplasmic contents in double membrane vesicles and deliver them to the lysosome for degradation. Autophagy plays critical regulatory roles in cancer, aging, neurodegeneration, immunity and many other physiological processes. Autophagy can be induced by multiple conditions such as starvation, viral infection, exercise and oxidative stress. In this work, we studied the role of autophagy in starvation-induced cell cycle arrest and quiescence entry, and studied the function and regulation of a major phosphorylation site of Beclin 1, serine 90. In the first study, we found that the in response to starvation, autophagy-deficient yeasts failed to arrest properly in G1/G0, but arrested in telophase with a quiescent-specific phenotype. In a second study, we found that Beclin 1 serine 90 is a major phosphorylation site of Beclin 1, which is induced by multiple stresses such as starvation and osmotic stress. The phosphorylation of Beclin 1 serine 90 leads to the activation of autophagy and inhibition of tumorigenesis. We identified MK2/3 as kinases that positively regulate autophagy by phosphorylating Beclin 1 at amino acid residue serine 90. We also found that Beclin 1 serine 90 phosphorylation is negatively regulated by Bcl-2 and positively regulated by AMPK. Beclin 1 serine 90 phosphorylation is also important for cell survival during high osmotic stress. Taken together, these results suggest that Beclin 1 serine 90 phosphorylation is a critical event in autophagy induction, which is tightly regulated by multiple kinases and regulatory proteins.Item Revealing Regulation and Organization of Signaling Networks by Scaffolding Proteins(2007-05-23) Swanik, Jackie Thomas; White, Michael A.Understanding the molecular mechanism that controls how cells respond to their environment is of major biological significance. The RAS/RAF signal transduction pathway is a good model with which to investigate this question as the major catalytic components of the pathway have been identified and it has been shown to elicit a wide variety of cellular responses such as proliferation, migration, differentiation, and apoptosis. The pathway is a three-tiered MAPK kinase cascade consisting of RAF, MEK, and ERK. Although the pathway has been extensively studied, pathway regulation is not completely understood. Genetic studies aimed at understanding pathway regulation have identified candidate scaffolding proteins. We used transient loss of function analysis to assess the contribution of the scaffolding proteins, Suppressor of RAS-8 (Sur-8) and Kinase Suppressor of RAS (KSR), to ligand mediated RAS/RAF signal transduction pathway activation. We show that Sur-8 and KSR are integral components of the RAS/RAF signal transduction pathway in mammalian cells. In addition, they display ligand specific coupling in that Sur-8 is required for EGF induced MEK activation while KSR is involved in LPA mediated MEK activation. to ligand mediated RAS/RAF signal transduction pathway activation. We show that Sur-8 and KSR are integral components of the RAS/RAF signal transduction pathway in mammalian cells. In addition, they display ligand specific coupling in that Sur-8 is required for EGF induced MEK activation while KSR is involved in LPA mediated MEK activation. Investigation of the molecular mechanism of action of Sur-8 and KSR found that Sur-8 is required for both EGF induced RAF-1 and B-RAF activation while KSR is involved in EGF induced RAF-1 activation. Additionally, Sur-8 contributes to RAF-1 localization as well as being associated with a RAF-1 activating kinase. Futhermore, we found that KSR does not impact LPA induced MEK activation through either RAF-1 or B-RAF activation and even though it impacts EGF induced RAF-1 activation it is not a limiting component to EGF induced MEK activation. In this study, we show that a function of scaffolding proteins in the RAS/RAF signal transduction pathway is to contribute to ligand specific coupling of MEK/ERK to distinct stimuli.Item Structural Analysis of STE20-Related Proline-Alanine-Rich Kinase (SPAK)(2009-06-17) Juang, Yu-Chi; Cobb, Melanie H.Ste20-related proline-alanine-rich kinase (SPAK) is a kinase that regulates ion cotransporters including KCC, NKCC1, NKCC2 and NCC. Recently, SPAK was identified as a target regulated by the WNK (With No lysine (K)) family of protein kinases. Overexpression of WNK1 has been associated to a hereditary form of hypertension. The link between WNK-SPAK-cotransporters provides one of the functional pathways for WNK to regulate intracellular and extracellular salt and water balance, thus contribute to the control of blood pressure. My study focused on determining the crystal structure of SPAK and providing a molecular basis for understanding the catalytic and regulatory mechanisms of this enzyme. I have solved the crystal structure of a partially active form of SPAK 63-390 (T243D). The structure contains the kinase domain and part of the C-terminal PF1 domain with a mutation of the WNK phosphorylation site, Thr243, to aspartate to mimic the phosphorylated state. The structure reveals two dimer interfaces. One is from the unique activation loop-swapped structure. The other is formed between the N-terminal domain of one molecule and the C-terminal domain of the second molecule. Comparison the structure with unphosphorylated inactive OSR1 revealed significant conformational changes in the glycine-rich loop, helix aC, and the activation loop. A more striking feature is the reformation of the P+1 pocket in SPAK. The interaction is formed by residues from both molecules of the domain-swapped dimer supporting the conclusion that the domain-swapped dimer is a functional unit. As remodeling of the P+1 pocket is usually associated with kinase activation, the conformational change found between the crystallized forms of OSR1 and SPAK is thus believed to be a regulated event. The unique activation loop-swapped feature suggests a novel regulatory mechanism that could be used in kinases.Item The Study of Wnt Signaling Effector POP-1/TCF in C. Elegans Early Embryos(2005-04-29) Lo, Miao-Chia; Lin, RueylingIn C. elegans embryos, the combined Wnt/MAPK pathway polarizes the founder cell of mesendoderm, EMS blastomere, such that EMS produces two daughters with distinct developmental fates. The posterior daughter E, whose fate is specified by Wnt/MAPK, generates intestinal tissues (endoderm), whereas the anterior daughter MS generates pharynx and muscle cells (mesoderm). The downstream Wnt/MAPK effector POP-1 is asymmetrically localized in the nuclei of A-P sisters including the MS/E pair, with a higher level in the anterior cells. This phenomenon is called POP-1 nuclear asymmetry. The Wnt/MAPK signaling is required for POP-1 nuclear asymmetry. It is believed that POP-1 represses endoderm fate in MS and Wnt/MAPK allows endoderm fate in E by downregulating the nuclear level of POP-1. In this study, the potential mechanisms for POP-1 nuclear asymmetry are presented. POP-1 nuclear asymmetry requires a 14-3-3 protein PAR-5 and at least three POP-1 potential phosphorylation sites for the MAPK LIT-1. LIT-1 activity is required for both POP-1/PAR-5 interaction and phosphorylation of at least two of the three potential LIT-1 sites in vivo. Nuclear export is also required for POP-1 nuclear asymmetry. The nuclear level of LIT-1 is higher in the E blastomere, which is regulated by the upstream kinase and Wnt signaling. All together, I propose that in the E blastomere, Wnt/MAPK signaling promotes PAR-5-mediated nuclear export of POP-1, thereby lowering its nuclear level. In addition to this differential nuclear export mechanism, POP-1 nuclear asymmetry may also be regulated by differential protein degradation. This study also shows that POP-1 functions to activate a Wnt/MAPK-responsive gene, sdz-23, in the E blastomere. This challenged the commonly accepted model of Wnt/MAPK-induced gene expression in E, which is based upon the alleviation of the repressive activity of POP-1. The activation of sdz-23 in E requires the ᭣atenin binding domain of POP-1 and a low nuclear level of POP-1. These results suggest that Wnt/MAPK converts the repressor POP-1 into a transcriptional activator and therefore, the non-canonical Wnt signaling in C. elegans early embryos is found to regulate its downstream effector POP-1 in a more canonical way than previously realized.