Prostate cancer (PCa) preferentially metastasizes to bone, leading to complications including severe pain, fractures, spinal cord compression, bone marrow suppression, and a mortality of ∼70%. In spite of recent advances in chemo-, hormonal, and radiation therapies, bone-metastatic, castrate-resistant PCa is incurable. PCa is somewhat unique among the solid tumors in its tendency to produce osteoblastic lesions composed of hypermineralized bone with multiple layers of poorly organized type I collagen fibrils that have reduced mechanical strength. Many of the signaling pathways that control normal bone homeostasis are at play in pathologic PCa bone metastases, including the receptor activator of nuclear factor-κB/receptor activator of nuclear factor-κB ligand/osteoprotegerin system. A number of PCa-derived soluble factors have been shown to induce the dysfunctional osteoblastic phenotype. However, therapies directed at these osteoblastic-stimulating proteins have yielded disappointing clinical results to date. One of the soluble factors expressed by PCa cells, particularly in bone metastases, is prostatic acid phosphatase (PAP). Human PAP is a prostate epithelium-specific secretory protein that was the first tumor marker ever described. Biologically, PAP exhibits both phosphatase activity and ecto-5′-nucleotidase activity, generating extracellular phosphate and adenosine as the final products. Accumulating evidence indicates that PAP plays a causal role in the osteoblastic phenotype and aberrant bone mineralization seen in bone-metastatic, castrate-resistant PCa. Targeting PAP may represent a therapeutic approach to improve morbidity and mortality from PCa osteoblastic bone metastases.

Cyclooxygenase-2 (COX-2) is constitutively expressed and highly regulated in the thick ascending limb (TAL). As COX-2 inhibitors (Coxibs) increase COX-2 expression, we tested the hypothesis that a negative feedback mechanism involving PGE(2) EP3 receptors regulates COX-2 expression in the TAL. Sprague-Dawley rats were treated with a Coxib [celecoxib (20 mg·kg(-1)·day(-1)) or rofecoxib (10 mg·kg(-1)·day(-1))], with or without sulprostone (20 μg·kg(-1)·day(-1)). Sulprostone was given using two protocols, namely, previous to Coxib treatment (prevention effect; Sulp7-Coxib5 group) and 5 days after initiation of Coxib treatment (regression effect; Coxib10-Sulp5 group). Immunohistochemical and morphometric analysis revealed that the stained area for COX-2-positive TAL cells (μm(2)/field) increased in Coxib-treated rats (Sham: 412 ± 56.3, Coxib: 794 ± 153.3). The Coxib effect was inhibited when sulprostone was used in either the prevention (285 ± 56.9) or regression (345 ± 51.1) protocols. Western blot analysis revealed a 2.1 ± 0.3-fold increase in COX-2 protein expression in the Coxib-treated group, an effect abolished by sulprostone using either the prevention (1.2 ± 0.3-fold) or regression (0.6 ± 0.4-fold vs. control, P < 0.05) protocols. Similarly, the 6.4 ± 0.6-fold increase in COX-2 mRNA abundance induced by Coxibs (P < 0.05) was inhibited by sulprostone; prevention: 0.9 ± 0.3-fold (P < 0.05) and regression: 0.6 ± 0.1 (P < 0.05). Administration of a selective EP3 receptor antagonist, L-798106, also increased the area for COX-2-stained cells, COX-2 mRNA accumulation, and protein expression in the TAL. Collectively, the data suggest that COX-2 levels are regulated by a novel negative feedback loop mediated by PGE(2) acting on its EP3 receptor in the TAL.

We tested the hypothesis that inhibition of EP3 receptors enhances cyclooxygenase (COX)-2 expression in the thick ascending limb (TAL) induced by hypertonic stimuli. COX-2 protein expression in the outer medulla increased approximately twofold in mice given free access to 1% NaCl in the drinking water for 3 days. The increase was associated with an approximate threefold elevation in COX-2 mRNA accumulation and an increase in PGE2 production by isolated medullary (m)TAL tubules from 77.3 ± 8.4 to 165.7 ± 10.8 pg/mg protein. Moreover, administration of NS-398 abolished the increase in PGE2 production induced by 1% NaCl. EP3 receptor mRNA levels also increased approximately twofold in the outer medulla of mice that ingested 1% NaCl. The selective EP3 receptor antagonist L-798106 increased COX-2 mRNA by twofold in mTAL tubules, and the elevation in COX-2 protein induced by 1% NaCl increased an additional 50% in mice given L-798106. COX-2 mRNA in primary mTAL cells increased twofold in response to media made hypertonic by the addition of NaCl (400 mosmol/kg H2O). L-798106 increased COX-2 mRNA twofold in isotonic media and fourfold in cells exposed to 400 mosmol/kg H2O. PGE2 production by mTAL cells increased from 79.3 ± 4.6 to 286.7 ± 6.3 pg/mg protein after challenge with 400 mosmol/kg H2O and was inhibited in cells transiently transfected with a lentivirus short hairpin RNA construct targeting exon 5 of COX-2 to silence COX-2. Collectively, the data suggest that local hypertonicity in the mTAL is associated with an increase in COX-2 expression concomitant with elevated EP3 receptor expression, which limits COX-2 activity in this segment of the nephron.

The mechanisms by which prostanoids contribute to the maintenance of whole body water homeostasis are complex and not fully understood. The present study demonstrates that an EP3-dependent feedback mechanism contributes to the regulation of water homeostasis under high-salt conditions. Rats on a normal diet and tap water were placed in metabolic cages and given either sulprostone (20 μg·kg·day) or vehicle for 3 days to activate EP3 receptors in the thick ascending limb (TAL). Treatment was continued for another 3 days in rats given either 1% NaCl in the drinking water or tap water. Sulprostone decreased expression of cyclooxygenase 2 (COX-2) expression by ∼75% in TAL tubules from rats given 1% NaCl concomitant with a ∼60% inhibition of COX-2-dependent PGE levels in the kidney. Urine volume increased after ingestion of 1% NaCl but was reduced ∼40% by sulprostone. In contrast, the highly selective EP3 receptor antagonist L-798106 (100 μg·kg·day), which increased COX-2 expression and renal PGE production, increased urine volume in rats given 1% NaCl. Sulprostone increased expression of aquaporin-2 (AQP2) in the inner medullary collecting duct plasma membrane in association with an increase in phosphorylation at Ser269 and decrease in Ser261 phosphorylation; antagonism of EP3 with L-798106 reduced AQP2 expression. Thus, although acute activation of EP3 by PGE in the TAL and collecting duct inhibits the Na-K-2Cl cotransporter and AQP2 activity, respectively, chronic activation of EP3 in vivo limits the extent of COX-2-derived PGE synthesis, thereby mitigating the inhibitory effects of PGE on these transporters and decreasing urine volume.

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