Low Salt Delivery Triggers Autocrine Release of Prostaglandin E2 From the Aldosterone-Sensitive Distal Nephron in Familial Hyperkalemic Hypertension Mice

Aberrant activation of with-no-lysine kinase (WNK)-STE20/SPS1-related proline-alanine-rich protein kinase (SPAK) kinase signaling in the distal convoluted tubule (DCT) causes unbridled activation of the thiazide-sensitive sodium chloride cotransporter (NCC), leading to familial hyperkalemic hypertension (FHHt) in humans. Studies in FHHt mice engineered to constitutively activate SPAK specifically in the DCT (CA-SPAK mice) revealed maladaptive remodeling of the aldosterone sensitive distal nephron (ASDN), characterized by decrease in the potassium excretory channel, renal outer medullary potassium (ROMK), and epithelial sodium channel (ENaC), that contributes to the hyperkalemia. The mechanisms by which NCC activation in DCT promotes remodeling of connecting tubule (CNT) are unknown, but paracrine communication and reduced salt delivery to the ASDN have been suspected. Here, we explore the involvement of prostaglandin E2 (PGE2). We found that PGE2 and the terminal PGE2 synthase, mPGES1, are increased in kidney cortex of CA-SPAK mice, compared to control or SPAK KO mice. Hydrochlorothiazide (HCTZ) reduced PGE2 to control levels, indicating increased PGE2 synthesis is dependent on increased NCC activity. Immunolocalization studies revealed mPGES1 is selectively increased in the CNT of CA-SPAK mice, implicating low salt-delivery to ASDN as the trigger. Salt titration studies in an in vitro ASDN cell model, mouse CCD cell (mCCD-CL1), confirmed PGE2 synthesis is activated by low salt, and revealed that response is paralleled by induction of mPGES1 gene expression. Finally, inhibition of the PGE2 receptor, EP1, in CA-SPAK mice partially restored potassium homeostasis as it partially rescued ROMK protein abundance, but not ENaC. Together, these data indicate low sodium delivery to the ASDN activates PGE2 synthesis and this inhibits ROMK through autocrine activation of the EP1 receptor. These findings provide new insights into the mechanism by which activation of sodium transport in the DCT causes remodeling of the ASDN.

NFκB-Activated COX2/PGE2/EP4 Axis Controls the Magnitude and Selectivity of BCG-Induced Inflammation in Human Bladder Cancer Tissues

Bacillus Calmette-Guérin (BCG) is commonly used in the immunotherapy of bladder cancer (BlCa) but its effectiveness is limited to only a fraction of patients. To identify the factors that regulate the response of human BlCa tumor microenvironment (TME) to BCG, we used the ex vivo whole-tissue explant model. The levels of COX2 in the BCG-activated explants closely correlated with the local production of Treg- and MDSCS attractants and suppressive factors, while the baseline COX2 levels did not have predictive value. Accordingly, we observed that BCG induced high levels of MDSC- and Treg-attracting chemokines (CCL22, CXCL8, CXCL12) and suppressive factors (IDO1, IL-10, NOS2). These undesirable effects were associated with the nuclear translocation of phosphorylated NFκB, induction of COX2, the key enzyme controlling PGE2 synthesis, and elevation of a PGE2 receptor, EP4. While NFκB blockade suppressed both the desirable and undesirable components of BCG-driven inflammation, the inhibitors of PGE2 synthesis (Celecoxib or Indomethacin) or signaling (EP4-selective blocker, ARY-007), selectively eliminated the induction of MDSC/Treg attractants and immunosuppressive factors but enhanced the production of CTL attractants, CCL5, CXCL9 and CXCL10. PGE2 blockade allowed for the selectively enhanced migration of CTLs to the BCG-treated BlCa samples and eliminated the enhanced migration of Tregs. Since the balance between the CTLs and suppressive cells in the TME predicts the outcomes in patients with BlCa and other diseases, our data help to elucidate the mechanisms which limit the effectiveness of BCG therapies and identify new targets to enhance their therapeutic effects.

Apoptotic endometrial caspase-3 mediated phospholipase a2 activation, a critical component in programing uterine receptivity

The objective of this study was to determine the consequence of uterine apoptotic caspase-3 activation on day 1 post coitus (dpc) in the pregnant mouse. We previously demonstrated that during pregnancy uterine caspase-3 activation isolated to the myometrial compartment is largely non-apoptotic and controls uterine quiescence. In this study we determined that uterine caspase-3 activation on 1 dpc may play a critical role in regulating endometrial PGE2 synthesis though iPLA2 activation. These analyses provide novel insight into the molecular mechanisms that regulate previously reported increases in endometrial PGE2 synthesis in very early pregnancy, that act to enhance uterine receptivity.We have identified the site and impact of that uterine apoptotic caspase-3 activation utilizing uteri isolated from non-pregnant control animals at estrous and diestrous and from control pregnant mice at 1-19 dpc. In addition, uteri were isolated from non-ligated controls (GD), unilateral (UL) and bilateral ligated (BL) uterine horn mouse models at 1, 3 and 6 dpc. Uteri were examined for apoptotic indices, such as caspase-3 activation and TUNEL staining. Immunohistochemical analysis was performed to identify the site of apoptotic caspase-3 activation. The presence of the truncated form of phospholipase A2 (tiPLA2) was examined as a measure of apoptotic caspase-3 mediated iPLA2 activation.Our analysis determined that apoptotic caspase-3 and iPLA2 activation were limited to the endometrial compartments of the control and unilateral uteri on 1dpc and were not found in the bilateral ligated uterine horn on 3 or 6 dpc. Our data indicates that the presence of a conceptus on 1 dpc triggers an increase in endometrial apoptotic caspase-3 mediated iPLA2 activation. iPLA2 when activated causes the hydrolysis of fatty acids resulting in arachidonic acid release and production of PGE2, which in early pregnancy has been demonstrated to act in a leutoprotective manner, prolonging progesterone synthesis and promoting uterine receptivity.

Paeonol suppresses invasion, migration, and epithelial-to-mesenchymal transition in colorectal cancer cells through inhibition of COX-2 and PGE2

Purpose: To observe the effects of paeonol on the invasion and migration of LoVo colorectal cancer cells, and investigate its possible mechanisms. Materials and Methods: Cell transwell assay and wound-healing assay were applied, and the results suggested that paeonol could significantly inhibit the invasion and migration abilities of LoVo cells, which was associated with a reduction in COX-2 expression and PGE2 synthesis. Treatment with the selective COX-2 inhibitor, celecoxib, or transient transfection of colorectal cancer cells with COX-2 siRNA, also inhibited cell invasion and migration. Results: The invasion and migration capacity were evaluated in LoVo cells by transwell assay and wound-healing in vitro. Compared with the control group, the invasion cells through Matrigel and the wound-healing rate were significantly decreased after treated with paeonol for 24 h. Paeonol treatment downregulated the expression of MMP-9 and downregulated the COX-2 expression and PGE2 synthesis in LoVo cells. Paeonol could up-regulate the expression of epithelial marker E-cadherin while down-regulate the expressions of mesenchymal markers, Fibronectin and Vimentin. Paeonol inhibited PI3K-Akt and MAPK-ERK pathways in LoVo cells. Celecoxib treatment significantly decreased the cells penetrating the matrigel in a dose-dependent manner. siRNA knockdown of COX-2 leaded to inhibition of cell invasion in LoVo cells. Knockdown of COX-2 increased the expression of E-cadherin, whereas the expressions of Fibronectin and Vimentin were downregulated. Conclusion: Paeonol may inhibit PI3K-Akt and MAPK-ERK pathways through suppressing of the COX-2 expression and PGE2 synthesis, thus inhibiting the cell invasion, migration, and EMT in LoVo cells.

Characteristics of lipid droplets and the expression of proteins involved in lipolysis in the murine cervix during mid-pregnancy

Lipid droplets (LDs) are reservoirs of arachidonoyl lipids for prostaglandin (PG) E2 synthesis, and progesterone can stimulate PGE2 synthesis; however, the relationship between progesterone and LD metabolism in the murine cervix remains unclear. In the present study we examined LD distribution and changes in the expression of proteins involved in lipolysis and autophagy in the murine cervix during pregnancy, and compared the findings with those in dioestrous mice. During mid-pregnancy, LDs were predominantly distributed in the cervical epithelium. Electron microscopy revealed the transfer of numerous LDs from the basal to apical region in the luminal epithelium, marked catabolism of LDs, an elevated number of LDs and autophagosomes and a higher LD:mitochondrion size ratio in murine cervical epithelial cells (P<0.05). In addition, immunohistochemical and western blotting analyses showed significantly higher cAMP-dependent protein kinase, adipose triglyceride lipase and hormone-sensitive lipase expression, and a higher light chain 3 (LC3) II:LC3I ratio in the stroma and smooth muscles and, particularly, in murine cervical epithelial cells, during mid-pregnancy than late dioestrus. In conclusion, these results suggest that the enhanced lipolysis of LDs and autophagy in murine cervical tissues were closely related to pregnancy and were possibly controlled by progesterone because LD catabolism may be necessary for energy provision and PGE2 synthesis to maintain a closed pregnant cervix.

Activation of HSD11B1 in the bovine cumulus-oocyte complex during IVM and IVF

The bovine cumulus-oocyte complex (COC) is capable of converting cortisone, an inert glucocorticoid to active cortisol. This mechanism is mediated by 11β-hydroxysteroid oxidoreductase type 1 (HSD11B1), whose expression dramatically increases in the mature COC. In this study, we investigate the time course expression of HSD11B1 and the enzyme activity in the bovine COC undergoing maturation and fertilization in relation to key events taking place in the COC. Bovine COCs were subjected to in vitro maturation (IVM) and fertilization (IVF). The activities of HSD11B1 and HSD11B2, which mediates the opposite reaction, were measured using a radiometric conversion assay. In parallel studies, cumulus expansion, P4 production and the expression of genes associated with ovulation were measured. The reductive activity of HSD11B1 increased in the latter half of IVM and remained high during IVF, whereas the oxidative activity of HSD11B2 remained unchanged over both periods. Consequently, the net glucocorticoid metabolism in the bovine COC shifted from inactivation to activation around the time of ovulation and fertilization. The increase in HSD11B1 expression lagged behind that of P4 increase and cumulus expansion but ahead of the expressions of genes responsible for PGE2 synthesis. The reductive activity of HSD11B1 was well correlated with the cumulus expansion rate. This outcome indicates that the ability of the cumulus to activate glucocorticoids is related to its ability to synthesize hyaluronan. These results also indicate that the activation of HSD11B1 is an integral part of the sequential events taking place at the ovulation and fertilization in the bovine COC.