Insights into the role of PHLPP2/Akt/GSK3ß/Fyn kinase/Nrf2 trajectory in the reno-protective effect of rosuvastatin against colistin-induced acute kidney injury in rats.

OBJECTIVES: Oxidative stress-mediated colistin's nephrotoxicity is associated with the diminished activity of nuclear factor erythroid 2-related factor 2 (Nrf2) that is primarily correlated with cellular PH domain and leucine-rich repeat protein phosphatase (PHLPP2) levels. This study investigated the possible modulation of PHLPP2/protein kinase B (Akt) trajectory as a critical regulator of Nrf2 stability by rosuvastatin (RST) to guard against colistin-induced oxidative renal damage in rats. METHODS: Colistin (300,000 IU/kg/day; i.p.) was injected for 6 consecutive days, and rats were treated simultaneously with RST orally at 10 or 20 mg/kg. KEY FINDINGS: RST enhanced renal nuclear Nrf2 translocation as revealed by immunohistochemical staining to boost the renal antioxidants, superoxide dismutase (SOD) and reduced glutathione (GSH) along with a marked reduction in caspase-3. Accordingly, rats treated with RST showed significant restoration of normal renal function and histological features. On the molecular level, RST effectively decreased the mRNA expression of PHLPP2 to promote Akt phosphorylation. Consequently, it deactivated GSK-3ß and reduced the gene expression of Fyn kinase in renal tissues. CONCLUSIONS: RST could attenuate colistin-induced oxidative acute kidney injury via its suppressive effect on PHLPP2 to endorse Nrf2 activity through modulating Akt/GSK3 ß/Fyn kinase trajectory.

Attenuation of cartilage pathogenesis in osteoarthritis by blocking the phosphorylation of tyrosine kinase Fyn to ß-catenin, AZD0530.

OBJECTIVE: To observe the effect of AZD0530 on the progression of knee OA after blocking ß-catenin phosphorylation and then dormancy of the Wnt/ß pathway by tyrosine kinase Fyn. METHODS: The levels of Fyn, ß-catenin, p-ß-catenin (Tyr142), the chondrocyte positive marker Aggrecan, and the chondrocyte negative marker MMP13 were observed in human knee tibial plateau chondrocytes in vivo and in vitro. Different doses of AZD0530 were used to treat chondrocytes of the human OA tibial plateau chondrocytes in vitro, and the degree of chondrocyte degeneration was observed. Different doses of AZD0530 were intraarticularly injected into OA rats to observe the degree of tibial plateau cartilage degeneration. RESULTS: When OA occurred in human knee, the levels of tyrosine kinase Fyn,ß-catenin and p-ß-catenin (Tyr142) in chondrocytes increased significantly.The level of Aggrecan decreased and MMP13 increased in chondrocytes. The levels of ß-catenin, p-ß-catenin (Tyr142) and MMP13 in chondrocytes decreased, while the level of Aggrecan increased after AZD0530 was used to intervene chondrocytes in vitro, which was positively correlated with the dose of AZD0530. Intra-articular injection of AZD0530 obviously attenuated the degeneration of articular cartilage, which was positively correlated with the dose of AZD0530. CONCLUSION: The level of Fyn in chondrocytes of human knee tibial plateau increased significantly when OA occurred. AZD0530 can inhibit tyrosine kinase Fyn from ß-catenin phosphorylation, a key Wnt/ß pathway protein, and then inhibit Wnt/ß pathway levels in chondrocytes. This finding also suggests that disruption of the Wnt/ß pathway with AZD0530 provides chondral protection in rat posttraumatic OA.

Click-Chemistry Based High Throughput Screening Platform for Modulators of Ras Palmitoylation.

Palmitoylation is a widespread, reversible lipid modification that has been implicated in regulating a variety of cellular processes. Approximately one thousand proteins are annotated as being palmitoylated, and for some of these, including several oncogenes of the Ras and Src families, palmitoylation is indispensable for protein function. Despite this wealth of disease-relevant targets, there are currently few effective pharmacological tools to interfere with protein palmitoylation. One reason for this lack of development is the dearth of assays to efficiently screen for small molecular inhibitors of palmitoylation. To address this shortcoming, we have developed a robust, high-throughput compatible, click chemistry-based approach to identify small molecules that interfere with the palmitoylation of Ras, a high value therapeutic target that is mutated in up to a third of human cancers. This assay design shows excellent performance in 384-well format and is sensitive to known, non-specific palmitoylation inhibitors. Further, we demonstrate an ideal counter-screening strategy, which relies on a target peptide from an unrelated protein, the Src-family kinase Fyn. The screening approach described here provides an integrated platform to identify specific modulators of palmitoylated proteins, demonstrated here for Ras and Fyn, but potentially applicable to pharmaceutical targets involved in a variety of human diseases.

Control of interneuron dendritic growth through NRG1/erbB4-mediated kalirin-7 disinhibition.

Neuregulin 1 (NRG1) is a secreted trophic factor that activates the postsynaptic erbB4 receptor tyrosine kinase. Both NRG1 and erbB4 have been repeatedly associated with schizophrenia, but their downstream targets are not well characterized. ErbB4 is highly abundant in interneurons, and NRG1-mediated erbB4 activation has been shown to modulate interneuron function, but the role for NRG1-erbB4 signaling in regulating interneuron dendritic growth is not well understood. Here we show that NRG1/erbB4 promote the growth of dendrites in mature interneurons through kalirin, a major dendritic Rac1-GEF. Recent studies have shown associations of the KALRN gene with schizophrenia. Our data point to an essential role of phosphorylation in kalirin-7's C terminus as the critical site for these effects. As reduced interneuron dendrite length occurs in schizophrenia, understanding how NRG1-erbB4 signaling modulates interneuron dendritic morphogenesis might shed light on disease-related alterations in cortical circuits.

Chromium(VI) stimulates Fyn to initiate innate immune gene induction in human airway epithelial cells.

Mechanisms for pathogenic metal signaling in airway injury or disease promotion are poorly understood. It is widely believed that one mechanism for pathogenic and possible carcinogenic effects of inhaled chromium (Cr(VI)) is inhibition of inducible gene transactivation. However, we recently reported that Cr(VI) inhibition of Sp1-dependent transactivation required signal transducer and activator of transcription 1 (STAT1)-dependent expression of an inhibitory protein in airway epithelium. Thus, Cr(VI) exposures can induce genes, and we hypothesized that this induction resulted from Cr(VI) signaling through an innate immune-like STAT1-dependent pathway initiated by Fyn. Exposure of human airway epithelial (BEAS-2B) cells to Cr(VI) selectively transactivated the STAT-responsive interferon-stimulated response element (ISRE) and induced ISRE-driven transactivation of interferon regulatory factor 7 (IRF7), without affecting the gamma interferon-activated site (GAS)-driven IRF1 expression. Cr(VI)-induced IRF7 was absent or greatly reduced in cells that lacked STAT1, were treated with the Src family kinase inhibitor, PP2, or lacked Fyn. Expressing Fyn, but not Src, in mouse embryonic fibroblasts cells null for Src, Yes, and Fyn restored Cr(VI)-stimulated STAT1 tyrosine phosphorylation and IRF7 expression. Finally, shRNA knockdown of Fyn in BEAS-2B cells prevented Cr(VI)-activated STAT1 transactivation of IRF7. These data support a novel mechanism through which Cr(VI) stimulates Fyn to initiate interferon-like signaling for STAT1-dependent gene transactivation.

Characterization of Fyn signaling on the age-dependent immuno-modulation on traumatic rats.

Traumatic stress is well characterized to develop immuno-depression in our previous report. Here, we provide evidence that adult and aged rats showed similar decrease in lymphocyte proliferation and natural killer (NK) cell activity. However, compared with beginning recovering from traumatic stress after 3 day and fully recovered by 7 day in adult rats, aged rats begin the recovery phage later than 3 day and do not fully recovered by 7 day. In parallel, Fyn expression in cerebral cortex was augmented with the highest level at 3 day of trauma in both age groups of rats, although aged rats exhibited lower level than the younger cohorts. Immune consequences were consequently modified by intracerebroventricular (i.c.v.) injection of Fyn antibody or recombinant adenovirus expressing active Fyn. Finally, the increase in Fyn expression was converged on ERK1/2 (extracellular signal regulated kinase 1/2) activation. Taken together, the data indicated that immunological processes in response to traumatic stress was age dependent, Fyn-ERK1/2 signal pathway was required to convey the recovery signals.

Fyn regulates eosinophil infiltration into the conjunctiva by downregulating the Th2 response.

PURPOSE: Under certain circumstances, fyn may serve to negatively regulate the differentiation of naïve helper T (Th) cells into Th2 cells. This study aimed to investigate whether fyn negatively regulates the development of experimental immune-mediated blepharoconjunctivitis (EC), in which Th2 cells play an important role in C57BL/6 mice. METHODS: C57BL/6 background wild-type (WT) or fyn knockout (fyn-/-) mice were subcutaneously immunized with ragweed (RW) adsorbed in aluminum hydroxide. Ten days later the mice were challenged with RW in eye drops, and 24 h after challenge, eyes, blood and spleens were harvested for histology, measurement of serum IgE, and proliferation or cytokine assays, respectively. RW-primed splenocytes from WT and fyn-/- mice were cultured in the presence of RW. Seventy-two hours later, either whole splenocytes or isolated CD4+T cells were transferred into syngeneic WT mice. Four days after the transfer, the recipient mice were challenged with RW and evaluated as described above. RESULTS: Infiltration of eosinophils into the conjunctiva induced by active immunization was significantly increased in fyn-/- mice relative to WT mice. Total serum IgE was also significantly higher in fyn-/- mice than in WT mice. In parallel, a higher level of IL-4 production from splenocytes was induced by concanavalin A stimulation in fyn-/- mice than in WT mice. In contrast to active immunization, transfer of whole splenocytes or separated CD4+T cells derived from WT or fyn-/- mice induced similar levels of eosinophilic infiltration in WT mice. CONCLUSIONS: Fyn regulates infiltration of eosinophils into the conjunctiva through downregulation of Th2 responses. This negative regulation is exerted only during the induction phase of EC.