Phenotypic Characterization of the Endocannabinoid-Degrading Enzyme Alpha/Beta-Hydrolase Domain 6 Knockout Rat.

Introduction: Alpha/beta-hydrolase domain 6 (ABHD6) is an enzyme that hydrolyzes 2-arachidonoylglycerol, a high-efficiency endogenous cannabinoid. Although the endocannabinoid system has been suggested to be involved in regulation of bladder function, the roles of ABHD6 in the control of micturition remain unknown. To elucidate the physiological and pathological roles of ABHD6 in vivo, we examined phenotypes of ABHD6 knockout rats (Abhd6-/-) generated by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins system. Materials and Methods: Age-matched knockout and wild-type (WT) rats of both sexes were used. Results: Expression of ABHD6, assessed by quantitative real-time polymerase chain reaction and Western blot analysis, was clearly diminished in Abhd6-/- rats compared with WT rats. Mutant rats had a normal appearance, and the body weight and food consumption were similar to those of WT rats. The interval between bladder contractions assessed by continuous cystometry was significantly shorter in ABHD6 knockout rats than in WT rats when the bladder was stimulated with acetic acid. Mechanical paw withdrawal thresholds measured by von Frey testing were significantly lowered in the knockout rats than in WT rats. The plasma levels of prostaglandin E2 (PGE2) and the stable metabolite of PGE2 in Abhd6-/- rats were twice as high as that in WT rats. Conclusions: Deletion of the ABHD6 gene in rats causes more frequent urination in the stimulated bladder and hyperalgesia to non-noxious mechanical stimuli along with increased plasma PGE2.

Roles of Pyk2 in signal transduction after gonadotropin-releasing hormone receptor stimulation.

The receptor for gonadotropin-releasing hormone (GnRH) is highly expressed in hypothalamic neurons. It has been reported that GnRH treatment of cultured GnRH neurons (GT1-7 cells) activated proline-rich tyrosine kinase 2 (Pyk2), and Pyk2 was involved in the activation of extracellular signal-regulated protein kinase 1 (ERK1) and ERK2 (ERK1/2). In the present study, we first examined the possibility that GnRH treatment might activate epidermal growth factor receptor (EGFR). We found that activation of EGFR after GnRH treatment for 5 min was much less than after EGF or heparin-binding EGF treatment. Next, we examined whether or not Pyk2 bound to growth factor receptor-binding protein 2 (Grb2). We overexpressed FLAG-fused Pyk2 in GT1-7 cells, and immunoprecipitated Pyk2 using an anti-FLAG antibody. The binding of Pyk2 to Grb2 was detected only after GnRH treatment. In contrast, a site-directed mutant of Pyk2 wherein tyrosine 881 was mutated to phenylalanine did not bind to Grb2. Studies with small interfering RNA and inhibitors indicated that the activation of Grb2/Ras/Raf/MEK was a major pathway to ERK1/2 activation after the short-term treatment of GT1-7 cells with GnRH.

Fyn-mediated phosphorylation of Pyk2 promotes its activation and dissociation downstream of gonadotropin-releasing hormone receptor.

The receptor for gonadotropin-releasing hormone (GnRH) is highly expressed in hypothalamic GnRH neurons, as well as in anterior pituitary gonadotrophs. In our previous study, we found that stimulation of the GnRH receptor activated protein kinase D1 (PKD1), and PKD1 was involved in the Fyn-mediated activation of proline-rich tyrosine kinase 2 (Pyk2) in cultured GnRH neurons (GT1-7 cells). In the present study, we examined the molecular mechanisms of Pyk2 activation and the interaction of Pyk2 and Fyn in GT1-7 cells. Experiments with site-directed mutants of Pyk2 indicated that tyrosine 402 (Tyr402) was phosphorylated both by autophosphorylation and by Fyn, whereas Tyr579 was phosphorylated mainly by Fyn. We found that dasatinib, a Src family inhibitor, enhanced the interaction of Pyk2 and Fyn. Experiments with site-directed mutants of Pyk2 and Fyn indicated that dasatinib enhanced the binding of Pyk2 autophosphorylated at Tyr402 and the Src homology 2 domain in Fyn. Our present data may suggest that fully activated Pyk2 dissociates from Fyn after Fyn-mediated phosphorylation of Pyk2 at sites other than Tyr402 and Tyr579.

Activation of Pyk2 by CaM kinase II in cultured hypothalamic neurons and gonadotroph cells.

Gonadotropin-releasing hormone (GnRH) is secreted from hypothalamic GnRH neurons and stimulates a GnRH receptor in gonadotroph cells and GnRH neurons. The GnRH receptor belongs to the G-protein-coupled receptors, and stimulation of the GnRH receptor activates extracellular signal-regulated protein kinase (ERK). We reported previously that the δ2 isoform of Ca2+ /calmodulin-dependent protein kinase II (CaM kinase IIδ2) was involved in GnRH-induced ERK activation in cultured GnRH neurons (GT1-7 cells). Recently, we found that GnRH treatment of GT1-7 cells activated proline-rich tyrosine kinase 2 (Pyk2), and Pyk2 was involved in ERK activation. In the current study, we examined the possibility that CaM kinase IIδ2 might activate Pyk2. Knockdown of CaM kinase IIδ2 and KN93, an inhibitor of CaM kinases, inhibited the GnRH-induced activation of Pyk2. In the case of cultured gonadotroph cells (αT3-1 cells), knockdown of CaM kinase IIß'e inhibited GnRH-induced Pyk2 activation. In addition, our inhibitor studies indicated that Pyk2 and CaM kinase II were involved in the GnRH-induced shedding of proHB-EGF in GT1-7 cells. These results suggested that CaM kinase II activated the ERK pathway through Pyk2 activation and HB-EGF production in response to GnRH.