Kinases of the Focal Adhesion Complex Contribute to Cardiomyocyte Specification.

Differentiation of pluripotent stem cells to cardiomyocytes is influenced by culture conditions including the extracellular matrices or similar synthetic scaffolds on which they are grown. However, the molecular mechanisms that link the scaffold with differentiation outcomes are not fully known. Here, we determined by immunofluorescence staining and mass spectrometry approaches that extracellular matrix (ECM) engagement by mouse pluripotent stem cells activates critical components of canonical wingless/integrated (Wnt) signaling pathways via kinases of the focal adhesion to drive cardiomyogenesis. These kinases were found to be differentially activated depending on type of ECM engaged. These outcomes begin to explain how varied ECM composition of in vivo tissues with development and in vitro model systems gives rise to different mature cell types, having broad practical applicability for the design of engineered tissues.

Disruption of anchoring junctions in the testes of experimental varicocele rats.

Varicocele is a common disease of the male reproductive system and is the main cause of male infertility; however, the pathological mechanisms of varicocele remain unclear. The anchoring junctions (AJs) in the testies are located between Sertoli cells, or between Sertoli cells and germ cells. Intact and functional AJs are crucial for spermatogenesis. In the present study, the histomorphology, ultrastructure of AJ, cell cycle, expression of AJ structural proteins, and the level of AJ-associated signaling molecules were investigated in the left testes of experimental varicocele rats at 8 and 12 weeks after surgery. The results revealed that varicocele induced the loss of premature germ cells from the seminiferous epithelium. Furthermore, the results of the present study also revealed damage to the AJ ultrastructure, disorientation of the spermatid head, deregulation of the cell cycle, downregulation of AJ structural proteins, enhanced phosphorylation of focal adhesion kinase (FAK) at Tyr397 and its downstream adapter Src at Tyr416, and activation of the extracellular signal-regulated protein kinase 1 (ERK1) signaling pathway. Thus, the present study demonstrated that varicocele disrupted the structure and function of AJs in the left testes of rats, and that enhancement of FAK phosphorylation may contribute to AJ damage by activating ERK1 signaling, disrupting actin-based filament networks, and altering the balance of the apical ectoplasmic specialization-blood testis barrier functional axis. These findings provide important insights into the pathological mechanisms through which varicocele contributes to male infertility and could help to identify new therapeutic targets for varicocele.

Phytochemicals Targeting VEGF and VEGF-Related Multifactors as Anticancer Therapy.

The role of vascular endothelial growth factor (VEGF) in cancer cells is not limited to angiogenesis; there are also multiple factors, such as neuropilins (non-tyrosine kinases receptors), tyrosine kinases receptors, immunodeficiencies, and integrins, that interact with VEGF signaling and cause cancer initiation. By combating these factors, tumor progression can be inhibited or limited. Natural products are sources of several bioactive phytochemicals that can interact with VEGF-promoting factors and inhibit them through various signaling pathways, thereby inhibiting cancer growth. This review provides a deeper understanding of the relation and interaction of VEGF with cancer-promoting factors and phytochemicals in order to develop multi-targeted cancer prevention and treatment.

Emerging targets in cancer drug resistance.

Drug resistance is a complex phenomenon that frequently develops as a failure to chemotherapy during cancer treatment. Malignant cells increasingly generate resistance to various chemotherapeutic drugs through distinct mechanisms and pathways. Understanding the molecular mechanisms involved in drug resistance remains an important area of research for identification of precise targets and drug discovery to improve therapeutic outcomes. This review highlights the role of some recent emerging targets and pathways which play critical role in driving drug resistance.

Effects of digitoxin on cell migration in ovarian cancer inflammatory microenvironment.

Clinical and experimental evidence supports a role for cardiac glycosides (CGs) as potential novel anticancer drugs. However, there are no studies reporting the effect of CGs on the inflammatory tumor microenvironment (TME), which plays a central role in tumor progression and invasiveness. We investigated whether digitoxin affects a) specific pathways involved in motility and/or activation of different cell types shaping TME, and b) cancer cell growth and invasiveness in response to TME-associated factors. To test our hypothesis, conditioned media (CM) from polarized macrophages, and apoptotic or non-apoptotic ovarian cancer cells (SKOV3) were tested as chemoattractants for endothelial cells, monocytes and cancer cells. We demonstrated that CM from M1 (LPS/IFNγ) and M2 (IL-4/IL-13) polarized macrophages, which mimic inflammatory TME, increased both HUVEC migration and tubularization. Treatment of HUVECs with digitoxin at concentrations within its plasma therapeutic range counteracted these effects. Digitoxin affected the expression of neither M1 (CD80/CD68) nor M2 (CD206/CD163) activation markers, nor the amount of cell-bound IL-1ß and CCL22. Accordingly, HUVEC migration in response to CM from digitoxin-treated activated macrophages was unchanged. These data point to a direct effect of digitoxin on HUVEC signaling rather than on the modulation of the cytokine profile released from activated macrophages. At variance with what observed for HUVECs, digitoxin did not prevent monocyte migration induced by SKOV3 CM. In addition, digitoxin significantly impaired SKOV3 growth and migration in response to M1 or M2 macrophage CM. Finally, we showed that digitoxin inhibited FAK phosphorylation in SKOV3 but not PYK2 phosphorylation in monocytes, thus providing a molecular mechanism accounting for the observed differential anti-migratory effect. Overall, digitoxin counteracted salient features of the inflammatory ovarian cancer microenvironment, laying the ground for potential digitoxin repositioning as an anticancer drug.

Imipramine administration induces changes in the phosphorylation of FAK and PYK2 and modulates signaling pathways related to their activity.

BACKGROUND: Antidepressants can modify neuronal functioning by affecting many levels of signal transduction pathways that are involved in neuroplasticity. We investigated whether the phosphorylation status of focal adhesion kinase (FAK/PTK2) and its homolog, PYK2/PTK2B, and their complex with the downstream effectors (Src kinase, p130Cas, and paxillin) are affected by administration of the antidepressant drug, imipramine. The treatment influence on the levels of ERK1/2 kinases and their phosphorylated forms (pERK1/2) or the Gαq, Gα11 and Gα12 proteins were also assessed. METHODS: Rats were injected with imipramine (10 mg/kg, twice daily) for 21 days. The levels of proteins investigated in their prefrontal cortices were measured by Western blotting. RESULTS: Imipramine induced contrasting changes in the phosphorylation of FAK and PYK2 at Tyr397 and Tyr402, respectively. The decreased FAK phosphorylation and increased PYK2 phosphorylation were reflected by changes in the levels of their complex with Src and p130Cas, which was observed predominantly after chronic imipramine treatment. Similarly only chronic imipramine decreased the Gαq expression while Gα11 and Gα12 proteins were untouched. Acute and chronic treatment with imipramine elevated ERK1 and ERK2 total protein levels, whereas only the pERK1 was significantly affected by the drug. CONCLUSION: The enhanced activation of PYK2 observed here could function as compensation for FAK inhibition. GENERAL SIGNIFICANCE: These data demonstrate that treatment with imipramine, which is a routine in counteracting depressive disorders, enhances the phosphorylation of PYK2, a non-receptor kinase instrumental in promoting synaptic plasticity. This effect documents as yet not considered target in the mechanism of imipramine action.

Microgravity influences circadian clock oscillation in human keratinocytes.

Microgravity and sudden changes of gravitational forces exert numerous effects on tissues, organs and apparatus. Responses to these forces variably applied to cells indicate the existence of mechanotransduction pathways able to modulate transcription. Oscillation of circadian clocks similarly influences many cellular and metabolic processes. Here we hypothesized that signals derived from changes of gravitational forces applied to epidermal cells might influence their physiology in harmony with the oscillation of the molecular clock. In this study, we describe amplified oscillations of Bmal1 circadian clock gene in human keratinocytes exposed to short simulated microgravity and to rapid variation of gravitational forces. We found that exposure to microgravity enhances the amplitude of the Bmal1 feedback loop sustained by an apparently lower variability of Rev-erbα transcription, while recovery from microgravity is characterized by increased amplitude of Bmal1 expression and elongation of the oscillatory periods of Bmal1 and Rev-erbα. These data highlight the existence of integrated signaling network connecting mechanosensitive pathways to circadian gene regulation.

Elucidation of the roles of the Src kinases in pancreatic acinar cell signaling.

Recent studies report the Src-family kinases (SFK's) are important in a number of physiological and pathophysiological responses of pancreatic acinar cells (pancreatitis, growth, apoptosis); however, the role of SFKs in various signaling cascades important in mediating these cell functions is either not investigated or unclear. To address this we investigated the action of SFKs in these signaling cascades in rat pancreatic acini by modulating SFK activity using three methods: adenovirus-induced expression of an inactive dominant-negative CSK (Dn-CSK-Advirus) or wild-type CSK (Wt-CSK-Advirus), which activate or inhibit SFK, respectively, or using the chemical inhibitor, PP2, with its inactive control, PP3. CCK (0.3, 100 nM) and TPA (1 µM) activated SFK and altered the activation of FAK proteins (PYK2, p125(FAK)), adaptor proteins (p130(CAS), paxillin), MAPK (p42/44, JNK, p38), Shc, PKC (PKD, MARCKS), Akt but not GSK3-ß. Changes in SFK activity by using the three methods of altering SFK activity affected CCK/TPAs activation of SFK, PYK2, p125(FAK), p130(CAS), Shc, paxillin, Akt but not p42/44, JNK, p38, PKC (PKD, MARCKS) or GSK3-ß. With chemical inhibition the active SFK inhibitor, PP2, but not the inactive control analogue, PP3, showed these effects. For all stimulated changes pre-incubation with both adenoviruses showed similar effects to chemical inhibition of SFK activity. In conclusion, using three different approaches to altering Src activity allowed us to define fully for the first time the roles of SFKs in acinar cell signaling. Our results show that in pancreatic acinar cells, SFKs play a much wider role than previously reported in activating a number of important cellular signaling cascades shown to be important in mediating both acinar cell physiological and pathophysiological responses.

Role of focal adhesion kinases in adhesion and migration of endothelial cells / 医用生物力学

Objective To investigate the effect of focal adhesion kinases (FAK) inhibitor with different concentration on the adhesion and migration of endothelial cells (ECs) and the expression of downstream Rac1 protein, and to explore the role of FAK in adhesion and migration of ECs by using FAK inhibitor to inhibit the phosphorylation of Y397 site of FAK. Method Scratch wound migration assay was performed to examine the effect of FAK inhibitor with different concentration (from 0 nmol/mL to 250 nmol/mL) on ECs migration at 2, 4, 8 and 24 h, respectively. Western blot combined with immunofluorescence analysis were performed to determine the effect of FAK inhibitor with different concentration on distribution and expression of Rac1 protein. Results With the concentration of FAK inhibitor increased, ECs migration distance and the Rac1 protein expression decreased. Conclusions The inhibition of FAK phosphorylation could inhibit cell adhesion and migration with the decrease in downstream Rac1 protein, and ECs adhesion/migration was related to FAK Rho GTPases signaling pathways.

Correlation between neck lymph node metastasis and focal adhesion kinases expression in oral squamous cell carcinoma / 实用口腔医学杂志

Objective: To investigate the correlation between neck lymph node metastasis and focal adhesion kinases(FAK) expression in oral squamous cell carcinoma(OSCC). Methods: Immunohistochemistry LsAB technique was used to observe the expression of FAK in primary tumour and nodal metastasis of OSCC in 80 cases, correlation between the expression of FAK in OSCC and neck lymph node metastasis was analyzed statistically. Results: The results demonstrated that FAK was expressed in all 80 cases, the expression of FAK in the peripheral area of OSCC was stronger than that in central area (P