Receptor Protein Tyrosine Phosphatase α-Mediated Enhancement of Rheumatoid Synovial Fibroblast Signaling and Promotion of Arthritis in Mice.

OBJECTIVE: During rheumatoid arthritis (RA), fibroblast-like synoviocytes (FLS) critically promote disease pathogenesis by aggressively invading the extracellular matrix of the joint. The focal adhesion kinase (FAK) signaling pathway is emerging as a contributor to the anomalous behavior of RA FLS. The receptor protein tyrosine phosphatase α (RPTPα), which is encoded by the PTPRA gene, is a key promoter of FAK signaling. The aim of this study was to investigate whether RPTPα mediates FLS aggressiveness and RA pathogenesis. METHODS: Through RPTPα knockdown, we assessed FLS gene expression by quantitative polymerase chain reaction analysis and enzyme-linked immunosorbent assay, invasion and migration by Transwell assays, survival by annexin V and propidium iodide staining, adhesion and spreading by immunofluorescence microscopy, and activation of signaling pathways by Western blotting of FLS lysates. Arthritis development was examined in RPTPα-knockout (KO) mice using the K/BxN serum-transfer model. The contribution of radiosensitive and radioresistant cells to disease was evaluated by reciprocal bone marrow transplantation. RESULTS: RPTPα was enriched in the RA synovial lining. RPTPα knockdown impaired RA FLS survival, spreading, migration, invasiveness, and responsiveness to platelet-derived growth factor, tumor necrosis factor, and interleukin-1 stimulation. These phenotypes correlated with increased phosphorylation of Src on inhibitory Y(527) and decreased phosphorylation of FAK on stimulatory Y(397) . Treatment of RA FLS with an inhibitor of FAK phenocopied the knockdown of RPTPα. RPTPα-KO mice were protected from arthritis development, which was due to radioresistant cells. CONCLUSION: By regulating the phosphorylation of Src and FAK, RPTPα mediates proinflammatory and proinvasive signaling in RA FLS, correlating with the promotion of disease in an FLS-dependent model of RA.

A novel mechanism of action for salidroside to alleviate diabetic albuminuria: effects on albumin transcytosis across glomerular endothelial cells.

Salidroside (SAL) is a phenylethanoid glycoside isolated from the medicinal plant Rhodiola rosea. R. rosea has been reported to have beneficial effects on diabetic nephropathy (DN) and high-glucose (HG)-induced mesangial cell proliferation. Given the importance of caveolin-1 (Cav-1) in transcytosis of albumin across the endothelial barrier, the present study was designed to elucidate whether SAL could inhibit Cav-1 phosphorylation and reduce the albumin transcytosis across glomerular endothelial cells (GECs) to alleviate diabetic albuminuria as well as to explore its upstream signaling pathway. To assess the therapeutic potential of SAL and the mechanisms involved in DN albuminuria, we orally administered SAL to db/db mice, and the effect of SAL on the albuminuria was measured. The albumin transcytosis across GECs was explored in a newly established in vitro cellular model. The ratio of albumin to creatinine was significantly reduced upon SAL treatment in db/db mice. SAL decreased the albumin transcytosis across GECs in both normoglycemic and hyperglycemic conditions. SAL reversed the HG-induced downregulation of AMP-activated protein kinase and upregulation of Src kinase and blocked the upregulation Cav-1 phosphorylation. Meanwhile, SAL decreased mitochondrial superoxide anion production and moderately depolarized mitochondrial membrane potential. We conclude that SAL exerts its proteinuria-alleviating effects by downregulation of Cav-1 phosphorylation and inhibition of albumin transcytosis across GECs. These studies provide the first evidence of interference with albumin transcytosis across GECs as a novel approach to the treatment of diabetic albuminuria.

Glabridin inhibits migration, invasion, and angiogenesis of human non-small cell lung cancer A549 cells by inhibiting the FAK/rho signaling pathway.

This study reports the antimigration, anti-invasive effect of glabridin, a flavonoid obtained from licorice, in human non-small cell lung cancer A549 cells. Glabridin exhibited effective inhibition of cell metastasis by decreasing cancer cell migration and invasion of A549 cells. In addition, glabridin also decreased A549-mediated angiogenesis. Further investigation revealed that glabridin's inhibition of cancer angiogenesis was also evident in a nude mice model. Blockade of A549 cells migration was associated with an increase of ανß3 integrin proteosome degradation. Glabridin also decreased the active forms of FAK and Src, and enhanced levels of inactivated phosphorylated Src (Tyr 527), decreasing the interaction of FAK and Src. Inhibition of the FAK/Src complex by glabridin also blocked Akt activation, resulting in reduced activation of RhoA and myosin light chain phosphorylation. This study demonstrates that glabridin may be a novel anticancer agent for the treatment of lung cancer in 3 different ways: inhibition of migration, invasion, and angiogenesis.

5-deoxykaempferol plays a potential therapeutic role by targeting multiple signaling pathways in skin cancer.

Nontoxic small molecules with multitargeting effects are believed to have potential in cancer prevention. Dietary phytochemicals were shown to exhibit cancer-preventive effects attributed to their antioxidant capacities. In this report, we show that the natural compound 5-deoxykaempferol (5-DK) exerts a chemopreventive effect on UVB-induced skin carcinogenesis by targeting multiple signaling molecules. 5-DK suppressed the UVB-induced expression of cyclooxygenase-2 (COX-2) and vascular endothelial growth factor in mouse skin epidermal JB6 P+ cells. Moreover, 5-DK inhibited phosphorylation of MKK3/6, MKK4, and Akt, but had no effect on phosphorylation of Src, extracellular signal-regulated kinases, or ribosomal S6 kinase (RSK). However, 5-DK affected multiple targets by reducing Src, phosphoinositide 3-kinase (PI3K), and RSK2 activities. In particular, pull-down assays revealed that 5-DK specifically bound to and competed with ATP for binding with Src, PI3K, and RSK2. Exposure to 5-DK significantly suppressed UVB-induced tumorigenesis in mouse skin in a dose-dependent manner, and it inhibited the UVB-induced expression of COX-2, proliferating cell nuclear antigen, vascular endothelial growth factor, and matrix metalloproteinase-9. Our data suggest that 5-DK docks at the ATP-binding site of Src, PI3K, and RSK2. For RSK2, the ATP-binding site is located between the N- and C-lobes of the kinase domain. Taken together, our results indicate that 5-DK holds promise for the treatment of UVB-induced skin cancer by targeting Src, PI3K, and RSK2 signaling.

Homozygous mutation of focal adhesion kinase in embryonic stem cell derived neurons: normal electrophysiological and morphological properties in vitro.

BACKGROUND: Genetically manipulated embryonic stem (ES) cell derived neurons (ESNs) provide a powerful system with which to study the consequences of gene manipulation in mature, synaptically connected neurons in vitro. Here we report a study of focal adhesion kinase (FAK), which has been implicated in synapse formation and regulation of ion channels, using the ESN system to circumvent the embryonic lethality of homozygous FAK mutant mice. RESULTS: Mouse ES cells carrying homozygous null mutations (FAK-/-) were generated and differentiated in vitro into neurons. FAK-/- ESNs extended axons and dendrites and formed morphologically and electrophysiologically intact synapses. A detailed study of NMDA receptor gated currents and voltage sensitive calcium currents revealed no difference in their magnitude, or modulation by tyrosine kinases. CONCLUSION: FAK does not have an obligatory role in neuronal differentiation, synapse formation or the expression of NMDA receptor or voltage-gated calcium currents under the conditions used in this study. The use of genetically modified ESNs has great potential for rapidly and effectively examining the consequences of neuronal gene manipulation and is complementary to mouse studies.