The ERK/MAP kinase pathway is an important regulator of gene expression and differentiation in postmitotic cells. To understand how this pathway controls gene expression in bone, we examined the subnuclear localization of P-ERK in differentiating osteoblasts. Induction of differentiation was accompanied by increased ERK phosphorylation and expression of osteoblast-related genes, including osteocalcin (Bglap2) and bone sialoprotein (Ibsp). Confocal immunofluorescence microscopy revealed that P-ERK colocalized with the RUNX2 transcription factor in the nuclei of differentiating cells. Interestingly, a portion of this nuclear P-ERK was directly bound to the proximal promoter regions of Bglap2 and Ibsp. Furthermore, the level of P-ERK binding to chromatin increased with differentiation, whereas RUNX2 binding remained relatively constant. The P-ERK-chromatin interaction was seen only in RUNX2-positive cells, required intact RUNX2-selective enhancer sequences, and was blocked with MAPK inhibition. These studies show for the first time that RUNX2 specifically targets P-ERK to the chromatin of osteoblast-related genes, where it may phosphorylate multiple substrates, including RUNX2, resulting in altered chromatin structure and gene expression.

Development of anticancer drugs targeting Aurora B, an important member of the serine/threonine kinases family, has been extensively focused on in recent years. In this work, by applying an integrated computational method, including comparative molecular field analysis (CoMFA), comparative molecular similarity indices analysis (CoMSIA), homology modeling and molecular docking, we investigated the structural determinants of Aurora B inhibitors based on three different series of derivatives of 108 molecules. The resultant optimum 3D-QSAR models exhibited (q(2) = 0.605, r(2) (pred) = 0.826), (q(2) = 0.52, r(2) (pred) = 0.798) and (q(2) = 0.582, r(2) (pred) = 0.971) for MK-0457, GSK1070916 and SNS-314 classes, respectively, and the 3D contour maps generated from these models were analyzed individually. The contour map analysis for the MK-0457 model revealed the relative importance of steric and electrostatic effects for Aurora B inhibition, whereas, the electronegative groups with hydrogen bond donating capacity showed a great impact on the inhibitory activity for the derivatives of GSK1070916. Additionally, the predictive model of the SNS-314 class revealed the great importance of hydrophobic favorable contour, since hydrophobic favorable substituents added to this region bind to a deep and narrow hydrophobic pocket composed of residues that are hydrophobic in nature and thus enhanced the inhibitory activity. Moreover, based on the docking study, a further comparison of the binding modes was accomplished to identify a set of critical residues that play a key role in stabilizing the drug-target interactions. Overall, the high level of consistency between the 3D contour maps and the topographical features of binding sites led to our identification of several key structural requirements for more potency inhibitors. Taken together, the results will serve as a basis for future drug development of inhibitors against Aurora B kinase for various tumors.

The planarian flatworm is an ideal model to study the regeneration due to its robust regenerative ability. A variety of cellular response activities have been reported to be involved in the regeneration process, including the mitogen-activated protein kinase (MAPK) signaling. However, the mechanism of MAPK pathway in regenerative responses is still unclear. In this study, we employed the planarian, Dugesia japonica, as the model to investigate the function of MAP-extracellular signal-regulated kinase (MEK), an important component of MAPK signaling pathway, in the regeneration process. We found that MEK was required for the missing tissue response after several amputation and subsequent regeneration. MEK not only affected the size of blastema in the early stage of regeneration by regulating stem cell proliferation, but also determined the planarian's regeneration through balancing cell proliferation and apoptosis. In addition, the activation of Wnt pathway partially rescued regenerative defects induced by inhibition of MEK. Taken together, our results highlight a crucial role of MEK signaling in the planarian regeneration.

Staphylococcus hyicus has caused great losses in the swine industry by inducing piglet exudative epidermitis (EE), sow mastitis, metritis, and other diseases and is a threat to human health. The pathogenesis of EE, sow mastitis, and metritis involves the interaction between the host and virulent protein factors of S. hyicus, however, the proteins that interact with the host, especially the host immune system, are unclear. In the present study, immunoproteomics was used to screen the immunogenic proteins of S. hyicus strain ZC-4. The cellular and secreted proteins of S. hyicus strain ZC-4 were obtained, separated by 2D gel electrophoresis, and further analyzed by western blot with S. hyicus strain ZC-4-infected swine serum. Finally, 28 specific immunogenic proteins including 15 cellular proteins and 13 secreted proteins, 26 of which were novel immunogenic proteins from S. hyicus, were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. To further verify their immunogenicity, two representative proteins (acetate kinase [cellular] and enolase [secreted]) were chosen for expression, and the resultant recombinant proteins could react with S. hyicus ZC-4-infected swine serum. In mice, both acetate kinase and enolase activated the immune response by increasing G-CSF and MCP-5 expression, and acetate kinase further activated the immune response by increasing IL-12 expression. Enolase can confer better protection against S.hycius than acetate kinase in mice. For the first time to our knowledge, our results provide detailed descriptions of the cellular and secreted proteins of S. hyicus strain ZC-4. These immunogenic proteins may contribute to investigation and elucidation of the pathogenesis of S. hyicus and provide new candidates for subunit vaccines in the future.

We sought to assess the effect of progestin on the apoptosis of epithelial ovarian cancer cell line SKOV-3 and via regulation of phosphorylation signaling in. Epithelial ovarian cancer cell line SKOV-3 was treated with medroxyprogestogen, phosphatidylinositol 3-kinase inhibitor LY294002 and vehicle control. Akt, phospho-Akt, Bcl-2 and phospho-Bad proteins were examined by immunoblotting assays. Medroxyprogestogen-induced apoptosis was assessed by MTT assays and Annexin V apoptosis assay. We found no significant difference in Akt and Bad expression in both the medroxyprogestogen groups and the control group. The levels of phospho-Akt, Bcl-2 and phospho-Bad were decreased in all the medroxyprogestogen groups and significantly decreased in the high dose mitogen-activated protein (MAP) group (10 µmol/L). Viability of SKOV-3 was reduced and apparent apoptosis of SKOV-3 cells was observed with increased doses of MAP. The findings suggest that medroxyprogestogen can induce SKOV-3 cell apoptosis by inhibiting Akt phosphorylation.

Toll-like receptor 4 (TLR4) has been implicated as a locus for initiation of paclitaxel related chemotherapy induced peripheral neuropathy (CIPN). This project explores the involvement of the immediate down-stream signal molecules in inducing paclitaxel CIPN. Mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NFκB) were measured in dorsal root ganglia (DRG) and the spinal cord over time using Western blot and immunohistochemistry in a rat model of paclitaxel CIPN. The effects of MAPK inhibitors in preventing and reversing behavioral signs of CIPN were also measured (group sizes 4-9). Extracellular signal related kinase (ERK1/2) and P38 but not c-Jun N terminal kinase (JNK) or PI3K-Akt signaling expression was increased in DRG. Phospho-ERK1/2 staining was co-localized to small CGRP-positive DRG neurons in cell profiles surrounding large DRG neurons consistent with satellite glial cells. The expression of phospho-P38 was co-localized to small IB4-positive and CGRP-positive DRG neurons. The TLR4 antagonist LPS derived from Rhodobacter sphaeroides (LPS-RS) inhibited paclitaxel-induced phosphorylation of ERK1/2 and P38. The MAPK inhibitors PD98059 (MEK1/2), U0126 (MEK1/2) and SB203580 (P38) prevented but did not reverse paclitaxel-induced behavioral hypersensitivity. Paclitaxel treatment resulted in phosphorylation of Inhibitor α of NFκB (IκBα) in DRG resulting in an apparent release of NFκB from the IκBα-NFκB complex as increased expression of nuclear NFκB was also observed. LPS-RS inhibited paclitaxel-induced translocation of NFκB in DRG. No change was observed in spinal NFκB. These results implicate TLR4 signaling via MAP kinases and NFκB in the induction and maintenance of paclitaxel-related CIPN.

Tonic inhibition mediated by extrasynaptic γ-aminobutyric acid type A receptors (GABAARs) critically regulates neuronal excitability and brain function. However, the mechanisms regulating tonic inhibition remain poorly understood. Here, we report that Shisa7 is critical for tonic inhibition regulation in hippocampal neurons. In juvenile Shisa7 knockout (KO) mice, α5-GABAAR-mediated tonic currents are significantly reduced. Mechanistically, Shisa7 is crucial for α5-GABAAR exocytosis. Additionally, Shisa7 regulation of tonic inhibition requires protein kinase A (PKA) that phosphorylates Shisa7 serine 405 (S405). Importantly, tonic inhibition undergoes activity-dependent regulation, and Shisa7 is required for homeostatic potentiation of tonic inhibition. Interestingly, in young adult Shisa7 KOs, basal tonic inhibition in hippocampal neurons is unaltered, largely due to the diminished α5-GABAAR component of tonic inhibition. However, at this stage, tonic inhibition oscillates during the daily sleep/wake cycle, a process requiring Shisa7. Together, these data demonstrate that intricate signaling mechanisms regulate tonic inhibition at different developmental stages and reveal a molecular link between sleep and tonic inhibition.

Lysophosphatidic acid (LPA), as a bioactive lipid, plays a variety of physiological and pathological roles via activating six types of G-protein-coupled LPA receptors (LPA1-6). Our preliminary study found that LPA1 is highly expressed in lung cancer tissues compared with paracancerous tissues, but the role of LPA1 in lung carcinoma is unclear. This study aimed to elucidate the association between LPA1 and lung tumour behaviour at the cellular and animal model levels. We found that LPA promoted the migration, proliferation and colony formation of a lung cancer cell line (A549). LPA1 and LPA3 are preferentially expressed in A549 cells, and both Ki16425 (LPA1 and LPA3 antagonist) and ono7300243 (LPA1 antagonist) completely blocked the LPA-induced actions. These results were further verified by experiments of the LPA1/3 overexpression and LPA1 knockdown A549 cells. Furthermore, LPA1 overexpression and knockdown A549 cells were used to assess the in vivo tumour-bearing animal model and the mechanism underlying LPA-induced actions. In the animal model, A549 cell-derived tumour volume was significantly increased by LPA1 overexpression and significantly decreased by LPA1 knockdown respectively, suggesting that LPA1 is a regulator of in vivo tumour formation. Our results also indicated that the LPA1/Gi/MAP kinase/NF-κB pathway is involved in LPA-induced oncogenic actions in A549 cells. Thus, targeting LPA1 may be a novel strategy for treating lung carcinoma.

Understanding the mechanisms underlying anti-tumor immunity is pivotal for improving immune-based cancer therapies. Here, we report that growth of BRAF-mutant melanoma cells is inhibited, up to complete rejection, in Siah2-/- mice. Growth-inhibited tumors exhibit increased numbers of intra-tumoral activated T cells and decreased expression of Ccl17, Ccl22, and Foxp3. Marked reduction in Treg proliferation and tumor infiltration coincide with G1 arrest in tumor infiltrated Siah2-/- Tregs in vivo or following T cell stimulation in culture, attributed to elevated expression of the cyclin-dependent kinase inhibitor p27, a Siah2 substrate. Growth of anti-PD-1 therapy resistant melanoma is effectively inhibited in Siah2-/- mice subjected to PD-1 blockade, indicating synergy between PD-1 blockade and Siah2 loss. Low SIAH2 and FOXP3 expression is identified in immune responsive human melanoma tumors. Overall, Siah2 regulation of Treg recruitment and cell cycle progression effectively controls melanoma development and Siah2 loss in the host sensitizes melanoma to anti-PD-1 therapy.

ARID1A is among the most commonly mutated tumor suppressor genes in hepatocellular carcinoma (HCC). In this study, we conduct a CRISPR-Cas9 synthetic lethality screen using ARID1A-deficient HCC cells to identify approaches to treat HCC patients harboring ARID1A deficiency. This strategy reveals that the survival of these ARID1A-deficient HCC cells is highly dependent on genes related to the tricarboxylic acid (TCA) cycle. Mechanistically, ARID1A loss represses expression of key glycolysis-related gene PKM, shifting cellular glucose metabolism from aerobic glycolysis to dependence on the TCA cycle and oxidative phosphorylation. Cuproptosis is a recently defined form of copper-induced cell death reported to directly target the TCA cycle. Here, we find that ARID1A-deficient HCC cells and xenograft tumors are highly sensitive to copper treatment. Together, these results offer evidence of the synthetic lethality between ARID1A deficiency and mitochondrial respiration impairment, suggesting that copper treatment constitutes a promising therapeutic strategy for selectively targeting ARID1A-deficient HCC.