Concept: Mitogen-activated protein kinase
LY2228820 dimesylate is a highly selective small molecule inhibitor of p38α and p38β mitogen activated protein kinases (MAPKs) that is currently under clinical investigation for human malignancies. p38 MAPK is implicated in a wide range of biological processes, in particular those that support tumorigenesis. One such process, angiogenesis, is required for tumor growth and metastasis, and many new cancer therapies are therefore directed against the tumor vasculature. Using an in vitro co-culture endothelial cord formation assay, a surrogate of angiogenesis, we investigated the role of p38 MAPK in growth factor and tumor-driven angiogenesis using LY2228820 dimesylate treatment and by shRNA gene knockdown. p38 MAPK was activated in endothelial cells upon growth factor stimulation with inhibition by LY2228820 dimesylate treatment causing a significant decrease in VEGF, bFGF, EGF, and IL-6 induced endothelial cord formation and an even more dramatic decrease in tumor-driven cord formation. In addition to involvement in downstream cytokine signaling, p38 MAPK was important for VEGF, bFGF, EGF, IL-6 and other proangiogenic cytokine secretion in stromal and tumor cells. LY2228820 dimesylate results were substantiated using p38α MAPK specific shRNA and shRNA against the downstream p38 MAPK effectors MAPKAPK-2 and HSP27. Using in vivo models of functional neoangiogenesis, LY2228820 dimesylate treatment reduced hemoglobin content in a plug assay and decreased VEGF-A stimulated vascularization in a mouse ear model. Thus, p38α MAPK is implicated in tumor angiogenesis through direct tumoral effects and through reduction of proangiogenic cytokine secretion via the microenvironment.
Distinct signaling pathways producing diverse cellular outcomes can utilize similar subsets of proteins. For example, proteins from the T cell receptor (TCR) early signaling complex (ESC) are also involved in interferon-α receptor signaling. Defining the mechanism for how these proteins function within a given pathway is important in understanding the integration and communication of signaling networks with one another. We investigated the contributions of the TCR ESC proteins Lck, ZAP-70, Vav1, SLP-76, and LAT to integrin outside-in signaling in human T cells. Lck, ZAP-70, SLP-76, Vav1, and LAT were activated by α4β1 outside-in signaling but in a manner different from TCR signaling. TCR stimulation recruits ESC proteins to activate the mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase (ERK). α4β1 outside-in-mediated ERK activation did not require TCR ESC proteins. However, α4β1 outside-in signaling induced CD25 and costimulated CD69 and this was dependent upon TCR ESC proteins. TCR and α4β1 outside-in signaling are integrated through the common use of TCR ESC proteins; however, these proteins display functionally distinct roles in these pathways. These novel insights into the crosstalk between integrin outside-in and TCR signaling pathways are highly relevant to the development of therapeutic strategies to overcome disease associated with T cell deregulation.
Heat-shock protein 70 (HSP70) is known to function as a protective molecular chaperone that is massively induced in response to misfolded proteins following cerebral ischemia. The objective of this study was to characterize HSP70 induction by Z-ligustilide and explore its potential role in protection against cerebral ischemia-reperfusion injury. Our results demonstrated that the intranasal administration of Z-ligustilide reduced infarct volume and improved neurological function in a rat stroke model. Meanwhile, Z-ligustilide enhanced the cell viability of PC12 cells insulted by oxygen glucose deprivation-reoxygenation (OGD-Reoxy) and decreased apoptotic and necrotic cell death. Importantly, Z-ligustilide induced HSP70 expression both in vitro and in vivo. Although heat-shock factor 1 (HSF1) nuclear translocation was promoted by Z-ligustilide, HSP70-based heat-shock element (HSE)-binding luciferase activity was not activated, and HSP70 expression responsive to Z-ligustilide was not attenuated by HSE decoy oligonucleotides. However, Z-ligustilide significantly activated the phosphorylation of mitogen-activated protein kinases (MAPKs). Further inhibition of MAPK activity by specific inhibitors attenuated HSP70 induction by Z-ligustilide. Meanwhile, downregulation of HSP70 using KNK437, an HSP70 synthesis inhibitor, or small hairpin RNA (shRNA) significantly attenuated the protection of Z-ligustilide against OGD-Reoxy-induced injury. Moreover, the application of specific inhibitors of MAPKs also achieved similar results. Finally, Z-ligustilide alleviated the accumulation of ubiquitinated proteins induced by OGD-Reoxy, which was inhibited by HSP70-shRNA. Taken together, our results demonstrated that Z-ligustilide may induce protective HSP70 expression via the activation of the MAPK pathway, but not canonical HSF1 transcription. HSP70 plays a key role in the protection of Z-ligustilide against OGD-Reoxy-induced injury.
Many patients with histiocytic disorders such as Langerhans cell histiocytosis (LCH) or Erdheim-Chester disease (ECD) have treatment-refractory disease or suffer recurrences. Recent findings of gene mutations in histiocytoses have generated options for targeted therapies. We sought to determine the utility of prospective sequencing of select genes to further characterize mutations and identify targeted therapies for patients with histiocytoses. Biopsies of 72 patients with a variety of histiocytoses underwent comprehensive genomic profiling with targeted DNA and RNA sequencing. Fifteen patients (21%) carried the known BRAF V600E mutation, and 11 patients (15%) carried various mutations in MAP2K1, which we confirm induce constitutive activation of extracellular signal-regulated kinase (ERK) and were sensitive to inhibitors of mitogen-activated protein kinase kinase (MEK, the product of MAP2K1). We also identified recurring ALK rearrangements, and 4 LCH patients with an uncommon in-frame deletion in BRAF (N486_P490del or N486_T491>K), resulting in constitutive activation of ERK with resistance to V600E-specific inhibitors. We subsequently describe clinical cases where patients with aggressive multisystem LCH experience dramatic and sustained responses to monotherapy with either dabrafenib or trametinib. These findings support our conclusion that comprehensive genomic profiling should be regularly applied to these disorders at diagnosis, and can positively impact clinical care.
Once melanomas have progressed with acquired resistance to mitogen-activated protein kinase (MAPK)-targeted therapy, mutational heterogeneity presents a major challenge. We therefore examined the therapy phase before acquired resistance had developed and discovered the melanoma survival oncogene MITF as a driver of an early non-mutational and reversible drug-tolerance state, which is induced by PAX3-mediated upregulation of MITF. A drug-repositioning screen identified the HIV1-protease inhibitor nelfinavir as potent suppressor of PAX3 and MITF expression. Nelfinavir profoundly sensitizes BRAF and NRAS mutant melanoma cells to MAPK-pathway inhibitors. Moreover, nelfinavir is effective in BRAF and NRAS mutant melanoma cells isolated from patients progressed on MAPK inhibitor (MAPKi) therapy and in BRAF/NRAS/PTEN mutant tumors. We demonstrate that inhibiting a driver of MAPKi-induced drug tolerance could improve current approaches of targeted melanoma therapy.
Kinase inhibition in the mitogen activated protein kinase (MAPK) pathway is a standard therapy for cancer patients with activating BRAF mutations. However, the anti-tumorigenic effect and clinical benefit are only transient, and tumors are prone to treatment resistance and relapse. To elucidate mechanistic insights into drug resistance, we have established an in vitro cellular model of MAPK inhibitor resistance in malignant melanoma.
The causative agent of toxoplasmosis, the intracellular parasite Toxoplasma gondii, delivers a protein, GRA24, into the cells it infects that interacts with the mitogen-activated protein (MAP) kinase p38α (MAPK14), leading to activation and nuclear translocation of the host kinase and a subsequent inflammatory response that controls the progress of the parasite. The purification of a recombinant complex of GRA24 and human p38α has allowed the molecular basis of this activation to be determined. GRA24 is shown to be intrinsically disordered, binding two kinases that act independently, and is the only factor required to bypass the canonical mitogen-activated protein kinase activation pathway. An adapted kinase interaction motif (KIM) forms a highly stable complex that competes with cytoplasmic regulatory partners. In addition, the recombinant complex forms a powerful in vitro tool to evaluate the specificity and effectiveness of p38α inhibitors that have advanced to clinical trials, as it provides a hitherto unavailable stable and highly active form of p38α.
ETHNOPHARMACOLOGICAL RELEVANCE: Taraxacumcoreanum Nakaiis a dandelion native to Korea and is widely consumed as an edible and medicinal herb. The aerial part of T. coreanum (TC) has been used therapeutically as a diuretic and anti-inflammatory agent, but its mechanism of action has not yet been evaluated. AIM OF THE STUDY: To investigate the anti-inflammatory potential of a TCchloroform fraction(TCC) and its mechanisms of action in vitro and in vivo. MATERIALS AND METHODS: Isolated mouse peritoneal macrophages were stimulated in vitro with interferon-γ (IFN-γ) and lipopolysaccharide (LPS) in the presence or absence of TCC. The anti-inflammatory effects of TCC were assessed by measuring nitric oxide (NO) and prostaglandin E(2) (PGE(2)) production, as well as expression of inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), IκBα, phospho-IKK, mitogen-activated protein kinases (MAPKs), and signal transducer and activator of transcription (STAT1). The effects of TCC were tested in vivo by measuring cytokine production and survival in a mouse model of lethal septic shock. And the standard compounds of TC were analyzed by HPLC using a C18 column. RESULTS: Treatment of primary macrophages with TCC in vitro significantly inhibited all of the inflammatory parameters measured, including LPS-induced NO and PGE(2) production, iNOS and COX-2 expression, IκBα degradation, IKK phosphorylation, and MAPK and STAT1 activation. In a mouse model of LPS-induced septic shock, TCC inhibited the production of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6, and increased survival by 83%.Standard compounds (gallic acid, syringic acid) of TC were qualified by HPLC analysis. CONCLUSIONS: TCC possesses potent anti-inflammatory activity in vitro and in vivo, which occurs at least partly through inhibition of proinflammatory signaling and mediator release. These results strongly support the therapeutic potential of TCC as an anti-inflammatory agent in vivo.
STUDY DESIGN.: An experimental comparative study on moderate epidural hypothermia (MEH) versus moderate systemic hypothermia (MSH) after spinal cord injury (SCI). OBJECTIVE.: To compare neuroprotective effects of hypothermia between MEH and MSH after SCI in rats. SUMMARY OF BACKGROUND DATA.: Experimental MEH or MSH has been attempted for neuroprotection after ischemic or traumatic SCI. However, there is no comparative study on neuroprotective effect of MEH and MSH after SCI. If hypothermia is to be considered as 1 modality for treating SCI, further studies on the advantages and disadvantages of hypothermia will be mandatory. METHODS.: A spinal cord contusion was produced in all 32 rats, and these rats were randomly divided into 4 groups-8 rats in each group: (1) the control group (spinal cord contusion only), (2) the methylprednisolone group, (3) the MEH group (28°C for 48 hr), and (4) the MSH group (32°C for 48 hr). The functional recovery was assessed using Basso, Beattie, Bresnahan scale and antiapoptotic and anti-inflammatory effects were assessed. RESULTS.: The Basso, Beattie, Bresnahan scale scores in both the hypothermia groups were significantly higher than that in the control group at 6 weeks. The numbers of terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells and OX-42 positive cells were significantly lower in both the MEH and MSH groups than that in the control group. The p38 mitogen-activated protein kinases expression of the treated groups was significantly lower than that of the control group. The expression of caspase-8 and caspase-9 significantly decreased in the treated groups compared with that of the control group. However, in terms of caspase-3, only the MSH group has shown to be significantly lower than that of the control group. CONCLUSION.: This study presented that both systemic and epidural hypothermia demonstrated neuroprotective effects after SCI. Systemic hypothermia showed more neuroprotective effect by antiapoptotic and anti-inflammatory effects.
The c-Jun N-terminal kinase (JNK) pathway forms part of the mitogen-activated protein kinase (MAPK) signaling pathways comprising a sequential three-tiered kinase cascade. Here, an upstream MAP3K (MEKK1) phosphorylates and activates a MAP2K (MKK4 and MKK7), which in turn phosphorylates and activates the MAPK, JNK. The C-terminal kinase domain of MEKK1 (MEKK-C) is constitutively active, while MKK4/7 and JNK are both activated by dual phosphorylation of S/Y, and T/Y residues within their activation loops, respectively. While improvements in the purification of large quantities of active JNKs have recently been made, inadequacies in their yield, purity, and the efficiency of their phosphorylation still exist. We describe a novel and robust method that further improves upon the purification of large yields of highly pure, phosphorylated JNK1β1, which is most suitable for biochemical and biophysical characterization. Codon harmonization of the JNK1β1 gene was used as a precautionary measure toward increasing the soluble overexpression of the kinase. While JNK1β1 and its substrate ATF2 were both purified to >99% purity as GST fusion proteins using GSH-agarose affinity chromatography and each cleaved from GST using thrombin, constitutively-active MEKK-C and inactive MKK4 were separately expressed in E. coli as thioredoxin-His(6)-tagged proteins and purified using urea refolding and Ni(2+)-IMAC, respectively. Activation of JNK1β1 was then achieved by successfully reconstituting the JNK MAPK activation cascade in vitro; MEKK-C was used to activate MKK4, which in turn was used to efficiently phosphorylate and activate large quantities of JNK1β1. Activated JNK1β1 was thereafter able to phosphorylate ATF2 with high catalytic efficiency.