Sphingosine kinase 2 deficiency increases proliferation and migration of renal mouse mesangial cells and fibroblasts

Both of the sphingosine kinase (SK) subtypes SK-1 and SK-2 catalyze the production of the bioactive lipid molecule sphingosine 1-phosphate (S1P). However, the subtype-specific cellular functions are largely unknown. In this study, we investigated the cellular function of SK-2 in primary mouse renal mesangial cells (mMC) and embryonic fibroblasts (MEF) from wild-type C57BL/6 or SK-2 knockout (SK2ko) mice. We found that SK2ko cells displayed a significantly higher proliferative and migratory activity when compared to wild-type cells, with concomitant increased cellular activities of the classical extracellular signal regulated kinase (ERK) and PI3K/Akt cascades, and of the small G protein RhoA. Furthermore, we detected an upregulation of SK-1 protein and S1P3 receptor mRNA expression in SK-2ko cells. The MEK inhibitor U0126 and the S1P1/3 receptor antagonist VPC23019 blocked the increased migration of SK-2ko cells. Additionally, S1P3ko mesangial cells showed a reduced proliferative behavior and reduced migration rate upon S1P stimulation, suggesting a crucial involvement of the S1P3 receptor. In summary, our data demonstrate that SK-2 exerts suppressive effects on cell growth and migration in renal mesangial cells and fibroblasts, and that therapeutic targeting of SKs for treating proliferative diseases requires subtype-selective inhibitors.

In vivo TCR Signaling in CD4(+) T Cells Imprints a Cell-Intrinsic, Transient Low-Motility Pattern Independent of Chemokine Receptor Expression Levels, or Microtubular Network, Integrin, and Protein Kinase C Activity

Intravital imaging has revealed that T cells change their migratory behavior during physiological activation inside lymphoid tissue. Yet, it remains less well investigated how the intrinsic migratory capacity of activated T cells is regulated by chemokine receptor levels or other regulatory elements. Here, we used an adjuvant-driven inflammation model to examine how motility patterns corresponded with CCR7, CXCR4, and CXCR5 expression levels on ovalbumin-specific DO11.10 CD4(+) T cells in draining lymph nodes. We found that while CCR7 and CXCR4 surface levels remained essentially unaltered during the first 48-72 h after activation of CD4(+) T cells, their in vitro chemokinetic and directed migratory capacity to the respective ligands, CCL19, CCL21, and CXCL12, was substantially reduced during this time window. Activated T cells recovered from this temporary decrease in motility on day 6 post immunization, coinciding with increased migration to the CXCR5 ligand CXCL13. The transiently impaired CD4(+) T cell motility pattern correlated with increased LFA-1 expression and augmented phosphorylation of the microtubule regulator Stathmin on day 3 post immunization, yet neither microtubule destabilization nor integrin blocking could reverse TCR-imprinted unresponsiveness. Furthermore, protein kinase C (PKC) inhibition did not restore chemotactic activity, ruling out PKC-mediated receptor desensitization as mechanism for reduced migration in activated T cells. Thus, we identify a cell-intrinsic, chemokine receptor level-uncoupled decrease in motility in CD4(+) T cells shortly after activation, coinciding with clonal expansion. The transiently reduced ability to react to chemokinetic and chemotactic stimuli may contribute to the sequestering of activated CD4(+) T cells in reactive peripheral lymph nodes, allowing for integration of costimulatory signals required for full activation.

Kinase signalling pathways in endometriosis: potential targets for non-hormonal therapeutics.

BACKGROUND Endometriosis, the growth of endometrial tissue outside the uterine cavity, is associated with chronic pelvic pain, subfertility and an increased risk of ovarian cancer. Current treatments include the surgical removal of the lesions or the induction of a hypoestrogenic state. However, a reappearance of the lesion after surgery is common and a hypoestrogenic state is less than optimal for women of reproductive age. Additional approaches are required. Endometriosis lesions exist in a unique microenvironment characterized by increased concentrations of hormones, inflammation, oxidative stress and iron. This environment influences cell survival through the binding of membrane receptors and a subsequent cascading activation of intracellular kinases that stimulate a cellular response. Many of these kinase signalling pathways are constitutively activated in endometriosis. These pathways are being investigated as therapeutic targets in other diseases and thus may also represent a target for endometriosis treatment. METHODS To identify relevant English language studies published up to 2015 on kinase signalling pathways in endometriosis, we searched the Pubmed database using the following search terms in various combinations; 'endometriosis', 'inflammation', 'oxidative stress', 'iron', 'kinase', 'NF kappa', 'mTOR', 'MAPK' 'p38', 'JNK', 'ERK' 'estrogen' and progesterone'. Further citing references were identified using the Scopus database and finally current clinical trials were searched on the clinicaltrials.gov trial registry. RESULTS The current literature on intracellular kinases activated by the endometriotic environment can be summarized into three main pathways that could be targeted for treatments: the canonical IKKβ/NFκB pathway, the MAPK pathways (ERK1/2, p38 and JNK) and the PI3K/AKT/mTOR pathway. A number of pharmaceutical compounds that target these pathways have been successfully trialled in in vitro and animal models of endometriosis, although they have not yet proceeded to clinical trials. The current generation of kinase inhibitors carry a potential for adverse side effects. CONCLUSIONS Kinase signalling pathways represent viable targets for endometriosis treatment. At present, however, further improvements in clinical efficacy and the profile of adverse effects are required before these compounds can be useful for long-term endometriosis treatment. A better understanding of the molecular activity of these kinases, including the specific extracellular compounds that lead to their activation in endometriotic cells specifically should facilitate their improvement and could potentially lead to new, non-hormonal treatments of endometriosis.

Neospora caninum calcium-dependent protein kinase 1 is an effective drug target for neosporosis therapy.

Despite the enormous economic importance of Neospora caninum related veterinary diseases, the number of effective therapeutic agents is relatively small. Development of new therapeutic strategies to combat the economic impact of neosporosis remains an important scientific endeavor. This study demonstrates molecular, structural and phenotypic evidence that N. caninum calcium-dependent protein kinase 1 (NcCDPK1) is a promising molecular target for neosporosis drug development. Recombinant NcCDPK1 was expressed, purified and screened against a select group of bumped kinase inhibitors (BKIs) previously shown to have low IC50s against Toxoplasma gondii CDPK1 and T. gondii tachyzoites. NcCDPK1 was inhibited by low concentrations of BKIs. The three-dimensional structure of NcCDPK1 in complex with BKIs was studied crystallographically. The BKI-NcCDPK1 structures demonstrated the structural basis for potency and selectivity. Calcium-dependent conformational changes in solution as characterized by small-angle X-ray scattering are consistent with previous structures in low Calcium-state but different in the Calcium-bound active state than predicted by X-ray crystallography. BKIs effectively inhibited N. caninum tachyzoite proliferation in vitro. Electron microscopic analysis of N. caninum cells revealed ultra-structural changes in the presence of BKI compound 1294. BKI compound 1294 interfered with an early step in Neospora tachyzoite host cell invasion and egress. Prolonged incubation in the presence of 1294 interfered produced observable interference with viability and replication. Oral dosing of BKI compound 1294 at 50 mg/kg for 5 days in established murine neosporosis resulted in a 10-fold reduced cerebral parasite burden compared to untreated control. Further experiments are needed to determine the PK, optimal dosage, and duration for effective treatment in cattle and dogs, but these data demonstrate proof-of-concept for BKIs, and 1294 specifically, for therapy of bovine and canine neosporosis.