P38 Kinase Research Articles

In silico and in vitro investigation of dual targeting Prima-1MET as precision therapeutic against lungs cancer

This study emphasizes the explorations of binding of Prima-1MET with two targets, p53 a tumor suppressor protein, and tyrosine kinase of epidermal growth factor receptor. In silico investigations reveal that Prima-1MET showed robust binding with both targets. Molecular docking simulations demonstrated the binding affinity of Prima-1MET with p53 and tyrosine kinase was found to be −38.601 kJ/mol and −38.976 kJ/mol. In addition, the stability of Prima-1MET was explored by molecular dynamics simulation. Prima-1MET attains stability in the binding site of the respective protein till the simulation period is over. Moreover, the free binding energy ΔG bind was calculated by the molecular mechanics Poisson Boltzmann surface area method. The ΔG bind of Prima-1MET with tyrosine kinase was found to be −58.585 ± 0.327 kJ/mol and with p53 it was −35.910 ± 0.335 kJ/mol. Next, cytotoxicity of the Prima-1MET was evaluated using multiple cancer cell lines and the IC50 value were ranging between 4.5 and 30 μM. The cell death was identified by apoptosis assay. Further, the p53 and tyrosine kinase expression was monitored using immunofluorescence techniques, it was found Prima-1MET induces the expression of p53 protein and mimics the level of tyrosine kinase oncogenic target. Also, reactive oxygen species (ROS) and membrane potential activity of Prima-1MET was evaluated by using a lung cancer cell line. A significant decrease in intracellular ROS was observed and resulted in disruption of mitochondrial transmembrane potential. This study uncovers the underlying mechanism of Prima-1MET and could be helpful to design further leads against lung cancers. Communicated by Ramaswamy H. Sarma

Hypolipidemic and Anti-Inflammatory Effects of Curcuma longa-Derived Bisacurone in High-Fat Diet-Fed Mice.

Turmeric (Curcuma longa) contains various compounds that potentially improve health. Bisacurone is a turmeric-derived compound but has been less studied compared to other compounds, such as curcumin. In this study, we aimed to evaluate the anti-inflammatory and lipid-lowering effects of bisacurone in high-fat diet (HFD)-fed mice. Mice were fed HFD to induce lipidemia and orally administered bisacurone daily for two weeks. Bisacurone reduced liver weight, serum cholesterol and triglyceride levels, and blood viscosity in mice. Splenocytes from bisacurone-treated mice produced lower levels of the pro-inflammatory cytokines IL-6 and TNF-α upon stimulation with a toll-like receptor (TLR) 4 ligand, lipopolysaccharide (LPS), and TLR1/2 ligand, Pam3CSK4, than those from untreated mice. Bisacurone also inhibited LPS-induced IL-6 and TNF-α production in the murine macrophage cell line, RAW264.7. Western blot analysis revealed that bisacurone inhibited the phosphorylation of IKKα/β and NF-κB p65 subunit, but not of the mitogen-activated protein kinases, p38 kinase and p42/44 kinases, and c-Jun N-terminal kinase in the cells. Collectively, these results suggest that bisacurone has the potential to reduce serum lipid levels and blood viscosity in mice with high-fat diet-induced lipidemia and modulate inflammation via inhibition of NF-κB-mediated pathways.

Silibinin Alleviates Lipopolysaccharide Induced Inflammation in Porcine Mammary Epithelial Cells via mTOR/NF-κB Signaling Pathway.

Inflammatory responses reduce milk production in lactating sow. Silymarin (Silibinin is main component) reduces the inflammatory reaction and increases milk yield in lactating sow in the previous study. In the present study, silibinin may be a previously unrecognized nutrients in inflammatory resolution in porcine mammary epithelial cells (PMECs) is hypothesized. PMECs are treated with or without lipopolysaccharide (LPS) in the absence or presence of silibinin to test cell viability, cell cycle, cell apoptosis, cellular inflammatory factors, and signaling protein phosphorylation and expression. Silibinin promotes the proliferation of PMEC independent of the estrogen pathway. In LPS-induced damage of PMECs, silibinin protects cell proliferation, as well as reduced cell apoptosis. Silibinin reverses the LPS-induced increase in tumor necrosis factor-alpha (TNF-α) expression compared with control. In addition, silibinin accentuates the LPS-induced decrease in the key proteins phosphorylated-ribosomal protein S6 (p-S6) and phosphorylated-mammalian target of rapamycin (p-mTOR) of the mammalian target of rapamycin (mTOR) signaling pathway. Furthermore, silibinin reverses the increase in phosphorylated-nuclear factor-kappa B p65 (p-NF-κB p65), phosphorylated-Ikappab-alpha (p-IκB-α), and phosphorylated-Mitogen-activated protein kinase p38 (p-MAPK p38) expression in LPS-induced damage in PMECs. This study highlights silibinin-mTOR/NF-κB axis plays an important role in the control of inflammation in PMECs, and suggests that silibinin may be an effective dietary strategy to alleviate the inflammatory response in lactating sow.

Dithiobiureas Palladium(II) complexes' studies: From their synthesis to their biological action

Dithiobiureas coordination chemistry towards palladium (II) ions and their possible application is presented and discussed. 1,6-(4-Methoxyphenyl)-2,5-dithiobiurea and 1,6-(4-chlorophenyl)-2,5-dithiobiurea afford two Pd(II) complexes with the general formula [Pd2(H2L)Cl2(PPh3)2]. The metal ion forms one chelate ring with the dithiobiurea, and binds to a triphenylphosphine and an additional leaving group cisplatin like. One of the complexes (1) is endowed not only with stability in DMSO and aqua solutions containing a biological buffer but also with cytotoxicity versus gastric cancer cell lines. Complex 1 does not interact covalently to DNA models, neither activates p53 or Checkpoint Kinase 1 key proteins for DNA damage response. Thus, we propose that complex 1 exerts its action by activating Mitogen-Activated Protein Kinases [p38, Extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinases (JNKs)] as cell death inductors.

Glial Glutamate Transporter Modulation Prevents Development of Complete Freund's Adjuvant-Induced Hyperalgesia and Allodynia in Mice.

Glial glutamate transporter (GLT-1) modulation in the hippocampus and anterior cingulate cortex (ACC) is critically involved in nociceptive pain. The objective of the study was to investigate the effects of 3-[[(2-methylphenyl) methyl] thio]-6-(2-pyridinyl)-pyridazine (LDN-212320), a GLT-1 activator, against microglial activation induced by complete Freund's adjuvant (CFA) in a mouse model of inflammatory pain. Furthermore, the effects of LDN-212320 on the protein expression of glial markers, such as ionized calcium-binding adaptor molecule 1 (Iba1), cluster of differentiation molecule 11b (CD11b), mitogen-activated protein kinases (p38), astroglial GLT-1, and connexin 43 (CX43), were measured in the hippocampus and ACC following CFA injection using the Western blot analysis and immunofluorescence assay. The effects of LDN-212320 on the pro-inflammatory cytokine interleukin-1β (IL-1β) in the hippocampus and ACC were also assessed using an enzyme-linked immunosorbent assay. Pretreatment with LDN-212320 (20 mg/kg) significantly reduced the CFA-induced tactile allodynia and thermal hyperalgesia. The anti-hyperalgesic and anti-allodynic effects of LDN-212320 were reversed by the GLT-1 antagonist DHK (10 mg/kg). Pretreatment with LDN-212320 significantly reduced CFA-induced microglial Iba1, CD11b, and p38 expression in the hippocampus and ACC. LDN-212320 markedly modulated astroglial GLT-1, CX43, and, IL-1β expression in the hippocampus and ACC. Overall, these results suggest that LDN-212320 prevents CFA-induced allodynia and hyperalgesia by upregulating astroglial GLT-1 and CX43 expression and decreasing microglial activation in the hippocampus and ACC. Therefore, LDN-212320 could be developed as a novel therapeutic drug candidate for chronic inflammatory pain.

Synthesis, In Silico Studies, and In Vitro Anti-Inflammatory Activity of Novel Imidazole Derivatives Targeting p38 MAP Kinase.

A series of eight novel N-substituted [4-(trifluoro methyl)-1H-imidazole-1-yl] amide derivatives (AA1-AA8) were synthesized, characterized, and evaluated for their in vitro p38 MAP kinase anti-inflammatory inhibitory activity. The synthesized compounds were obtained by coupling [4-(trifluoromethyl)-1H-imidazole-1-yl] acetic acid with 2-amino-N-(Substituted)-3-phenylpropanamide derivatives utilizing 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b] pyridinium 3-oxide hexafluorophosphate as a coupling agent. Various spectroscopic methods established and confirmed their structures, specifically, 1H NMR, 13C NMR, Fourier transform infrared (FTIR), and mass spectrometry. In order to emphasize the binding site of the p38 MAP kinase protein and newly synthesized compounds, molecular docking studies were carried out. In the series, compound AA6 had the highest docking score of 7.83 kcal/mol. The ADME studies were performed using web software. Studies revealed that all the synthesized compounds were orally active and showed good gastrointestinal absorption within the acceptable range. Lipinski's "rule of five" was used to determine drug-likeness. The synthesized compounds were screened for their anti-inflammatory activity by performing an albumin denaturation assay in which five compounds (AA2, AA3, AA4, AA5, and AA6) were found to exhibit substantial activity. Hence, these were further selected and proceeded for the evaluation of p38 MAP kinase inhibitory activity. The compound AA6 possesses considerable p38 kinase inhibitory anti-inflammatory activity with an IC50 value of 403.57 ± 6.35 nM compared to the prototype drug adezmapimod (SB203580) with an IC50 value of 222.44 ± 5.98 nM. Some further structural modifications in compound AA6 could contribute to the development of new p38 MAP kinase inhibitors with an improved IC50 value.

Fluorescence resonance energy transfer (FRET) spatiotemporal mapping of atypical P38 reveals an endosomal and cytosolic spatial bias

Mitogen-activated protein kinase (MAPK) p38 is a central regulator of intracellular signaling, driving physiological and pathological pathways. With over 150 downstream targets, it is predicted that spatial positioning and the availability of cofactors and substrates determines kinase signaling specificity. The subcellular localization of p38 is highly dynamic to facilitate the selective activation of spatially restricted substrates. However, the spatial dynamics of atypical p38 inflammatory signaling are understudied. We utilized subcellular targeted fluorescence resonance energy transfer (FRET) p38 activity biosensors to map the spatial profile of kinase activity. Through comparative analysis of plasma membrane, cytosolic, nuclear, and endosomal compartments, we confirm a characteristic profile of nuclear bias for mitogen-activated kinase kinase 3/6 (MKK3/6) dependent p38 activation. Conversely, atypical p38 activation via thrombin-mediated protease-activated receptor 1 (PAR1) activity led to enhanced p38 activity at the endosome and cytosol, limiting nuclear p38 activity, a profile conserved for prostaglandin E2 activation of p38. Conversely, perturbation of receptor endocytosis led to spatiotemporal switching of thrombin signaling, reducing endosomal and cytosolic p38 activity and increasing nuclear activity. The data presented reveal the spatiotemporal dynamics of p38 activity and provide critical insight into how atypical p38 signaling drives differential signaling responses through spatial sequestration of kinase activity.

Two-Pore-Domain Potassium Channel TREK-1 Mediates Pulmonary Fibrosis through Macrophage M2 Polarization and by Direct Promotion of Fibroblast Differentiation.

Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by myofibroblast proliferation and abnormal accumulation of extracellular matrix in the lungs. After lung injury, M2 macrophages mediate the pathogenesis of pulmonary fibrosis by secreting fibrotic cytokines that promote myofibroblast activation. The TWIK-related potassium channel (TREK-1, also known as KCNK2) is a K2P channel that is highly expressed in cardiac, lung, and other tissues; it worsens various tumors, such as ovarian cancer and prostate cancer, and mediates cardiac fibrosis. However, the role of TREK-1 in lung fibrosis remains unclear. This study aimed to examine the effects of TREK-1 on bleomycin (BLM)-induced lung fibrosis. The results show that TREK-1 knockdown, mediated by the adenovirus or pharmacological inhibition of TREK-1 with fluoxetine, resulted in diminished BLM-induced lung fibrosis. TREK-1 overexpression in macrophages remarkably increased the M2 phenotype, resulting in fibroblast activation. Furthermore, TREK-1 knockdown and fluoxetine administration directly reduced the differentiation of fibroblasts to myofibroblasts by inhibiting the focal adhesion kinase (FAK)/p38 mitogen-activated protein kinases (p38)/Yes-associated protein (YAP) signaling pathway. In conclusion, TREK-1 plays a central role in the pathogenesis of BLM-induced lung fibrosis, which serves as a theoretical basis for the inhibition of TREK-1 as a potential therapy protocol for lung fibrosis.

Inhibitory effect of fucoidan on TNF-α-induced inflammation in human retinal pigment epithelium cells.

Sargassum horneri (S. horneri) is a brown seaweed that contains a fucose-rich sulfated polysaccharide called fucoidan and is known to possess beneficial bioactivities, such as anti-inflammatory, antiviral, antioxidative, and antitumoral effects. This study aimed to determine the anti-inflammatory effects of AB_SH (hydrothermal extracts from S. horneri) and its bioactive compound (fucoidan) against tumor necrosis factor alpha (TNF-α)-induced inflammation in human retinal pigment epithelial (RPE) cells. AB_SH did not exhibit any cytotoxicity, and it decreased the mRNA expression of interleukin (IL)-6 and IL-8 and the production of the cytokines IL-6 and TNF-α. It also suppressed the expression levels of phosphorylated nuclear factor kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs), including c-Jun amino-terminal kinases (JNK), p38 protein kinases (p38), and extracellular signal-regulated kinase (ERK) proteins, suggesting that AB_SH inhibits activation of the NF-kB/MAPK signaling pathway. Since fucoidan was identified in the composition analysis of AB_SH, it was additionally shown to be required for its anti-inflammatory effects in TNF-α-stimulated human RPE cells. In line with the AB_SH results, fucoidan reduced the mRNA levels of IL-6, IL-1ß, and IL-8 and production of the cytokines IL-6, TNF-α, and IL-8 through the downregulation of the NF-kB/MAPK signaling pathway in a dose-dependent manner. Collectively, the ability of AB_SH from S. horneri hydrothermal extracts to reduce inflammation indicates that it may be a good functional ingredient for managing ocular disorders.

A tri-herb formulation protects against ethanol-induced mouse liver injury and downregulates mitogen-activated protein kinase phosphatase 1

BackgroundA tri-herb formulation comprising Ganoderma (the dried fruiting body of Ganoderma lucidum), Puerariae Thomsonii Radix (the dried root of Pueraria thomsonii) and Hoveniae Semen (the dried mature seed of Hovenia acerba) —GPH for short— has been using for treating liver injury; however, the pharmacological basis of this application of GPH is unknown. This study aimed to investigate the liver protective effects and mechanisms of action of an ethanolic extract of GPH (GPHE) in mice. MethodsTo control the quality of GPHE, the contents of ganodermanontriol, puerarin and kaempferol in the extract were quantified by ultra-performance liquid chromatography. An ethanol (6 ml/kg, i.g.)-induced liver injury ICR mouse model was employed to investigate the hepatoprotective effects of GPHE. RNA-sequencing analysis and bioassays were performed to reveal the mechanisms of action of GPHE. ResultsThe contents of ganodermanontriol, puerarin and kaempferol in GPHE were 0.0632%, 3.627% and 0.0149%, respectively. Daily i.g. administration of 0.25, 0.5 or 1 g/kg of GPHE for 15 consecutive days suppressed ethanol (6 ml/kg, i.g., at day 15)-induced upregulation of serum AST and ALT levels and improved histological conditions in mouse livers, indicating that GPHE protects mice from ethanol-induced liver injury. Mechanistically, GPHE downregulated the mRNA level of Dusp1 (encoding MKP1 protein, an inhibitor of the mitogen-activated protein kinases JNK, p38 and ERK), and upregulated expression and phosphorylation of JNK, p38 and ERK, which are involved in cell survival in mouse liver tissues. Also, GPHE increased PCNA (a cell proliferation marker) expression and reduced TUNEL-positive (apoptotic) cells in mouse livers. ConclusionGPHE protects against ethanol-induced liver injury, and this effect of GPHE is associated with regulation of the MKP1/MAPK pathway. This study provides pharmacological justifications for the use of GPH in treating liver injury, and suggests that GPHE has potential to be developed into a modern medication for managing liver injury.

Abstract 4804: p38 MAPK-dependent regulation of HIF-1 and expression of the hypoxia-inducible gene IGFBP-3 in leiomyosarcoma cells

Abstract Hypoxia is a common feature of tumor microenvironments and stimulates a variety of responses in a cell. Tumor hypoxia is also associated with poor prognosis in soft tissue sarcomas (STS), including leiomyosarcomas. Genes upregulated under hypoxia contribute to the adaptation of tumor cells and promote tumor progression. Using transcriptome-wide gene expression analysis, we identified IGFBP-3 as one of the most highly upregulated genes in SK-LMS-1 leiomyosarcoma cells in hypoxic conditions. IGFBP-3 has been studied in several tumor types, including STS, and is associated with their altered pathogenesis. Mechanisms responsible for the hypoxia-induced expression of IGFBP-3 in STS have not been previously well defined. Hypoxia results in the stabilization of the hypoxia-inducible transcription factor, HIF-1, and production of reactive oxygen species (ROS) in other cell types. Here, we examined HIF-1 and ROS-dependent mechanisms to determine their roles in the hypoxia-induced expression of IGFBP-3 and other genes in SK-LMS-1 cells. Our data indicate that both ROS production and HIF-1 activation are required for the enhanced IGFBP-3 mRNA expression in hypoxic SK-LMS-1 cells. We also tested the roles of the stress activated protein kinase p38 and the tumor suppressor p53 for their potential to contribute to the regulation of IGFBP-3 expression in these cells. Inhibition of p53 tumor suppressor activity only partially reduced IGFBP-3 expression. We also determined that neither hypoxia nor inhibition of p53 affected the expression of the p53 target gene, CDKN1A (p21), in these cells. In contrast, inhibition of p38 MAPK completely prevented the hypoxia-induced increase in IGFBP-3 expression. Additionally, we found that p38 MAPK inhibition reduced HIF-1 protein accumulation and activity but had no effect on its mRNA expression under hypoxic conditions. Together these results suggest that ROS-dependent activation of p38 MAPK stabilizes HIF-1 and promotes the downstream expression of IGFBP-3. In contrast, p53 appears to have a limited effect on the expression of IGFBP-3 in SK-LMS-1 cells. Our results clarify the signaling pathway involved in the response of soft tissue sarcoma cells to hypoxia and may provide new avenues for intervention in cancer progression. Citation Format: Nuha Haque, Eric Shelden. p38 MAPK-dependent regulation of HIF-1 and expression of the hypoxia-inducible gene IGFBP-3 in leiomyosarcoma cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4804.

Lindenane-Type Sesquiterpene Dimers Mitigate Lipopolysaccharide-Induced Inflammation by Inhibiting Toll-Like Receptor Signaling.

Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns and trigger an inflammatory response via the myeloid differential factor 88 (MyD88)-dependent and toll-interleukin-1 receptor domain-containing adapter-inducing interferon-β (TRIF)-dependent pathways. Lindenane type sesquiterpene dimers (LSDs) are characteristic metabolites of plants belonging to the genus Sarcandra (Chloranthaceae). The aim of this study was to evaluate the potential anti-inflammatory effects of the LSDs shizukaol D (1) and sarcandrolide E (2) on lipopolysaccharides (LPS)-stimulated RAW264.7 macrophages in vitro, and explore the underlying mechanisms. Both LSDs neutralized the LPS-induced morphological changes and production of nitric oxide (NO), as determined by CCK-8 assay and Griess assay, respectively. Furthermore, shizukaol D (1) and sarcandrolide E (2) downregulated interferon β (IFNβ), tumor necrosis factor α (TNFα) and interleukin-1β (IL-1β) mRNA levels as measured by reverse transcription polymerase chain reaction (RT-PCR), and inhibited the phosphorylation of nuclear factor kappa B p65 (p65), nuclear factor kappa-Bα (IκBα), Jun N-terminal kinase (JNK), extracellular regulated kinase (ERK), mitogen-activated protein kinase p38 (p38), MyD88, IL-1RI-associated protein kinase 1 (IRAK1), and transforming growth factor-β-activated kinase 1 (TAK1) proteins in the Western blotting assay. In conclusion, LSDs can alleviate the inflammatory response by inhibiting the TLR/MyD88 signalling pathway.

ISG20L1 acts as a co-activator of DAPK1 in the activation of the p53-dependent cell death pathway.

Our previous studies have revealed that GADD45α is a liable proapoptotic protein, which undergoes MDM2-dependent constitutive ubiquitylation and degradation in resting cancer cells. Under chemotherapeutic agent (such as arsenite, 5-Fu and VP-16) exposure, DAPK1 functions as a novel p53 (also known as TP53) kinase, which induces phosphorylation of p53 at Ser15 and transactivates the p53 target Ets-1, to synergistically repress IKKβ-dependent MDM2 stability, and ultimately removes the inhibitory effect of MDM2 on GADD45α, resulting in GADD45α accumulation and cell apoptosis. In the current study, we show that there is a strong induction of ISG20L1 (also known as AEN) expression in several cancer cell lines under exposure of arsenite and other chemotherapeutic agents. Surprisingly, although originally identified as a transcriptional target of p53, ISG20L1 induction was not controlled by p53. Instead, ISG20L1 functioned as upstream activator of p53 by interacting with DAPK1, and plays an essential role in promoting DAPK1-p53 complex formation and the subsequent activation of Ets-1/IKKβ/MDM2/GADD45α cascade. Therefore, our findings have revealed novel function of ISG20L1 in mediating cancer cell apoptosis induced by chemotherapeutic agents via modulating activation of the DAPK1- and p53-dependent cell death pathway.

The Effects of Neuropeptide B on Proliferation and Differentiation of Porcine White Preadipocytes into Mature Adipocytes

Neuropeptide B (NPB) affects energy homeostasis and metabolism by binding and activating NPBWR1 and NPBWR2 in humans and pigs. Recently, we reported that NPB promotes the adipogenesis of rat white and brown preadipocytes as well as 3T3-L1 cells. In the present study, we evaluated the effects of NPB on the proliferation and differentiation of white porcine preadipocytes into mature adipocytes. We identified the presence of NPB, NPBWR1, and NPBWR2 on the mRNA and protein levels in porcine white preadipocytes. During the differentiation process, NPB increased the mRNA expression of PPARγ, C/EBPβ, C/EBPα, PPARγ, and C/EBPβ protein production in porcine preadipocytes. Furthermore, NPB stimulated lipid accumulation in porcine preadipocytes. Moreover, NPB promoted the phosphorylation of the p38 kinase in porcine preadipocytes, but failed to induce ERK1/2 phosphorylation. NPB failed to stimulate the expression of C/EBPβ in the presence of the p38 inhibitor. Taken together, we report that NPB promotes the differentiation of porcine preadipocytes via a p38-dependent mechanism.

Computational and Functional Analysis of Structural Features in the ZAKα Kinase.

The kinase ZAKα acts as the proximal sensor of translational impairment and ribotoxic stress, which results in the activation of the MAP kinases p38 and JNK. Despite recent insights into the functions and binding partners of individual protein domains in ZAKα, the mechanisms by which ZAKα binds ribosomes and becomes activated have remained elusive. Here, we highlight a short, thrice-repeated, and positively charged peptide motif as critical for the ribotoxic stress-sensing function of the Sensor (S) domain of ZAKα. We use this insight to demonstrate that the mutation of the SAM domain uncouples ZAKα activity from ribosome binding. Finally, we use 3D structural comparison to identify and functionally characterize an additional folded domain in ZAKα with structural homology to YEATS domains. These insights allow us to formulate a model for ribosome-templated ZAKα activation based on the re-organization of interactions between modular protein domains. In sum, our work both advances our understanding of the protein domains and 3D architecture of the ZAKα kinase and furthers our understanding of how the ribotoxic stress response is activated.

E3 Ubiquitin Ligase Constitutive Photomorphogenic 1 Regulates Differentiation and Inflammation via MAPK Signaling Pathway in Rabbit Articular Chondrocytes

Constitutive photomorphogenic 1 (COP1), is an E3 ubiquitin ligase that plays a role in the regulation of various cellular processes including cell growth, differentiation, and survival in mammals. In certain conditions such as overexpression or loss of function, COP1 acts either as an oncogenic protein or as a tumor suppressor by targeting specific proteins for ubiquitination-mediated degradation. However, the precise role of COP1 has not been well studied in primary articular chondrocytes. In this study, we investigated the role of COP1 in chondrocyte differentiation. Western blotting and reverse transcription-polymerase chain reaction analysis demonstrated that COP1 overexpression reduced type II collagen expression, promoted cyclooxygenase 2 (COX-2) expression, and reduced sulfated proteoglycan synthesis, as detected by Alcian blue staining. Upon siRNA treatment, revived type II collagen, sulfated proteoglycan production, and decreased COX-2 expression. Phosphorylation of p38 kinase and ERK-1/-2 signaling pathways was regulated by COP1 upon cDNA and siRNA transfection in chondrocytes. The inhibition of the p38 kinase and ERK-1/-2 signaling pathways with SB203580 and PD98059 ameliorated the expression of type II collagen and COX-2 in transfected chondrocytes, thus suggesting that COP1 regulates differentiation and inflammation in rabbit articular chondrocytes via the p38 kinase and ERK-1/-2 signaling pathway.

The influence of a synthetic growth hormone-releasing hormone analogue G11 and opioid peptide biphalin on selected fibroblasts parameters relevant to wound healing.

The treatment of hard-to-heal chronic wounds is still a major medical problem and an economic and social burden. In this work, we examine the proregenerative potential of two peptides, G11 (a trypsin-resistant analogue of growth hormone-releasing hormone [GHRH]) and biphalin (opioid peptide), and their combination in vitro on human fibroblasts (BJ). G11, biphalin and their combination exhibited no toxicity against BJ cells. On the contrary, these treatments significantly stimulated proliferation and migration of fibroblasts. Under inflammatory conditions (LPS-induced BJ cells), we noticed that the tested peptides decreased the levels of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) and interleukin 1β (IL-1β). This was correlated with diminished phosphorylation levels of p38 kinase, but not those of ERK1/2. We found also that G11, biphalin and their combination activated the ERK1/2 signalling pathway, which has been previously implicated in promigratory activity of some regeneration enhancers, including opioids or GHRH analogues. Potential application of their combination requires further work, in particular in vivo experiments, in which the organism-level relevance of the discussed cell-level effects would be proven and, additionally, analgesic action of the opioid ingredient could be quantified.

Glyceraldehyde-3-phosphate dehydrogenase regulates activation of c-Jun N-terminal kinase under oxidative stress

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) acts as a sensor under oxidative stress, leading to induction of various biological responses. Given that mitogen-activated protein kinase (MAPK) signaling pathways mediate cellular responses to a wide variety of stimuli, including oxidative stress, here, we aimed to elucidate whether a cross-talk cascade between GAPDH and MAPKs occurs under oxidative stress. Of the three typical MAPKs investigated—extracellular signal-regulated kinase, p38, and c-Jun N-terminal kinase (JNK)—we found that hydrogen peroxide (H2O2)-induced JNK activation is significantly reduced in HEK293 cells treated with small-interfering (si)RNA targeting GAPDH. Co-immunoprecipitation with a GAPDH antibody further revealed protein–protein interactions between GAPDH and JNK in H2O2-stmulated cells. Notably, both JNK activation and these interactions depend on oxidation of the active-site cysteine (Cys152) in GAPDH, as demonstrated by rescue experiments with either exogenous wild-type GAPDH or the cysteine-substituted mutant (C152A) in endogenous GAPDH-knockdown HEK293 cells. Moreover, H2O2-induced translocation of Bcl-2-associated X protein (Bax) into mitochondria, which occurs downstream of JNK activation, is attenuated by endogenous GAPDH knockdown in HEK293 cells. These results suggest a novel role for GAPDH in the JNK signaling pathway under oxidative stress.

Loss of EPS8 sensitizes non-small-cell lung carcinoma to chemotherapy-induced DNA damage.

Epidermal growth factor receptor pathway substrate number 8 (EPS8) has been reported to be critical in mediating tumor progression. However, the molecular and biological consequences of EPS8 overexpression remain unclear. Here we evaluated whether EPS8 increased DNA damage repair in non-small-cell lung carcinoma (NSCLC) cells and the mechanism of EPS8-mediated DNA damage repair which influenced chemosensitivity. Serial studies of functional experiments revealed that EPS8 knockdown inhibited cell growth, induced cell-cycle arrest and increased cisplatin therapeutic effects on NSCLC. EPS8 was found to induce DNA damage repair via upregulation of phosphorylated-ATM and downregulation of the tumor suppressor p53 and G1 cell kinase inhibitor p21. Moreover, in conjunction with cisplatin, decreasing EPS8 protein levels further increased p53 protein level and inhibited ATM signaling. Transplanted tumor studies were also performed to demonstrate that EPS8 knockdown inhibited tumor growth and sensitized tumors to cisplatin treatment. In conclusion, we have described a novel molecular mechanism through which EPS8 is likely to be involved in cancer progression and chemoresistance via DNA damage repair, indicating that EPS8 expression may influence the response to chemotherapy. Therefore, targeting EPS8 may be a potential therapeutic approach for patients with NSCLC.

TLR2 and 4 signaling pathways are altered in macrophages from V30M TTR mice with down-regulated expression of chemokines.

Hereditary amyloid transthyretin (ATTRv) amyloidosis is a fatal neurodegenerative disorder, first identified in Portugal. The most common transthyretin (TTR) mutation in ATTRv results from an exchange of a methionine for a valine at position 30 (V30M). ATTRv is characterized by the extracellular deposition of aggregates and fibrils of mutant forms of TTR, particularly in the nerves and ganglia of the peripheral nervous system (PNS). This phenotype is often accompanied by the lack of inflammatory infiltrates, despite the importance of macrophages in removal of TTR deposits in ATTRv patients. The mechanisms underlying this impairment of inflammatory responses in ATTRv patients are poorly understood. Here, we show a significant down-regulation in the expression of several chemokines by bone marrow-derived macrophages (BMDM) generated from V30M TTR mice upon stimulation with toll-like receptor 4 (TLR4) and TLR2 agonists. The phosphorylation of the MAP kinase p38, important for TLR4 and TLR2 signaling pathways, was also down-regulated in V30M macrophages, as compared with wild-type (WT) ones. The present study contributes with new insights to unravel the molecular mechanisms underlying the lack of inflammatory immune responses observed in ATTRv patients and may help in the development of new immune therapeutic strategies for the disease.