Prostaglandin E2 as a therapeutic target in bladder cancer: From basic science to clinical trials.

Bladder cancer (BCa) is a common solid tumor marked by high rates of recurrence, especially in non-muscle invasive disease. Prostaglandin E2 (PGE2) is a ubiquitously present lipid mediator responsible for numerous physiological actions. Inhibition of cyclooxygenase (COX) enzymes by the non-steroidal anti-inflammatory (NSAID) class of drugs results in reduced PGE2 levels. NSAID usage has been associated with reductions in cancers such as BCa. Clinical trials using NSAIDs to prevent recurrence have had mixed results, but largely converge on issues with cardiotoxicity. The purpose of this review is to understand the basic science behind how and why inhibitors of PGE2 may be effective against BCa, and to explore alternate therapeutic modalities for addressing the role of PGE2 without the associated cardiotoxicity. We will address the role of PGE2 in a diverse array of cancer-related functions including stemness, immunosuppression, proliferation, cellular signaling and more.

Prostaglandins and Bone.

Prostaglandins (PGs) are highly bioactive fatty acids. PGs, especially prostaglandin E2 (PGE2), are abundantly produced by cells of both the bone-forming (osteoblast) lineage and the bone-resorbing (osteoclast) lineage. The inducible cyclooxygenase, COX-2, is largely responsible for most PGE2 production in bone, and once released, PGE2 is rapidly degraded in vivo. COX-2 is induced by multiple agonists - hormones, growth factors, and proinflammatory factors - and the resulting PGE2 may mediate, amplify, or, as we have recently shown for parathyroid hormone (PTH), inhibit responses to these agonists. In vitro, PGE2 can directly stimulate osteoblast differentiation and, indirectly via stimulation of RANKL in osteoblastic cells, stimulate the differentiation of osteoclasts. The net balance of these two effects of PGE2 in vivo on bone formation and bone resorption has been hard to predict and, as expected for such a widespread local factor, hard to study. Some of the complexity of PGE2 actions on bone can be explained by the fact that there are four receptors for PGE2 (EP1-4). Some of the major actions of PGE2 in vitro occur via EP2 and EP4, both of which can stimulate cAMP signaling, but there are other distinct signaling pathways, important in other tissues, which have not yet been fully elucidated in bone cells. Giving PGE2 or agonists of EP2 and EP4 to accelerate bone repair has been examined with positive results. Further studies to clarify the pathways of PGE2 action in bone may allow us to identify new and more effective ways to deliver the therapeutic benefits of PGE2 in skeletal disorders.

The Role of Pyruvate Dehydrogenase Kinase-4 (PDK4) in Bladder Cancer and Chemoresistance.

Advanced bladder cancer remains a major source of mortality, with poor treatment options. Cisplatin-based chemotherapy is the standard treatment, however many patients are or become resistant. One potential cause of chemoresistance is the Warburg effect, a metabolic switch to aerobic glycolysis that occurs in many cancers. Upregulation of the pyruvate dehydrogenase kinase family (PDK1-PDK4) is associated with aerobic glycolysis and chemoresistance through inhibition of the pyruvate dehydrogenase complex (PDH). We have previously observed upregulation of PDK4 in high-grade compared with low-grade bladder cancers. We initiated this study to determine if inhibition of PDK4 could reduce tumor growth rates or sensitize bladder cancer cells to cisplatin. Upregulation of PDK4 in malignant bladder cancer cell lines as compared with benign transformed urothelial cells was confirmed using qPCR. Inhibition of PDK4 with dichloroacetate (DCA) resulted in increased PDH activity, reduced cell growth, and G0-G1 phase arrest in bladder cancer cells. Similarly, siRNA knockdown of PDK4 inhibited bladder cancer cell proliferation. Cotreatment of bladder cancer cells with cisplatin and DCA did not increase caspase-3 activity but did enhance overall cell death in vitro Although daily treatment with 200 mg/kg DCA alone did not reduce tumor volumes in a xenograft model, combination treatment with cisplatin resulted in dramatically reduced tumor volumes as compared with either DCA or cisplatin alone. This was attributed to substantial intratumoral necrosis. These findings indicate inhibition of PDK4 may potentiate cisplatin-induced cell death and warrant further studies investigating the mechanism through which this occurs. Mol Cancer Ther; 17(9); 2004-12. ©2018 AACR.

Continuous PTH in Male Mice Causes Bone Loss Because It Induces Serum Amyloid A.

Increased bone resorption is considered to explain why intermittent PTH is anabolic for bone but continuous PTH is catabolic. However, when cyclooxygenase-2 (COX2) is absent in mice, continuous PTH becomes anabolic without decreased resorption. In murine bone marrow stromal cells (BMSCs), serum amyloid A (SAA)3, induced in the hematopoietic lineage by the combination of COX2-produced prostaglandin and receptor activator of nuclear factor κB ligand (RANKL), suppresses PTH-stimulated osteoblast differentiation. To determine whether SAA3 inhibits the anabolic effects of PTH in vivo, wild-type (WT) and SAA3 knockout (KO) mice were infused with PTH. In WT mice, continuous PTH induced SAA3 and was catabolic for bone. In KO mice, PTH was anabolic, increasing trabecular bone, serum markers of bone formation, and osteogenic gene expression. In contrast, PTH increased all measurements associated with bone resorption, as well as COX2 gene expression, similarly in KO and WT mice. SAA1 and SAA2 in humans are likely to have analogous functions to SAA3 in mice. RANKL induced both SAA1 and SAA2 in human bone marrow macrophages in a COX2-dependent manner. PTH stimulated osteogenesis in human BMSCs only when COX2 or RANKL was inhibited. Addition of recombinant SAA1 or SAA2 blocked PTH-stimulated osteogenesis. In summary, SAA3 suppresses the bone formation responses but not the bone resorption responses to PTH in mice, and in the absence of SAA3, continuous PTH is anabolic. In vitro studies in human bone marrow suggest that SAA may be a target for enhancing the therapeutic effects of PTH in treating osteoporosis.

SATB1 and bladder cancer: Is there a functional link?

PURPOSE: SATB1, a global genome organizer, has been shown to play a role in the development and progression of some solid tumors, but its role in bladder cancer is undetermined. Moreover, there is conflicting data about the role of SATB1 in other tumors. This study was initiated to assess a potential role for SATB1 with the hypothesis that SATB1 acts as a tumor promoter in bladder cancer. MATERIALS AND METHODS: We evaluated SATB1 expression in bladder cancer cell lines (HTB-5, HTB-9) and compared them to a benign urothelial cell line (UROtsa). Short-hairpin RNA was used to silence SATB1 in multiple cell lines, and cell death and cell proliferation were assessed using multiple assays. RESULTS: SATB1 expression was increased significantly in all cancer cell lines compared to benign urothelial cells. SATB1 expression was knocked down by short-hairpin RNA and functional outcomes, including cell number, cell-cycle arrest, cell viability, and apoptosis after cisplatin treatment, were measured. Surprisingly, knockdown of SATB1 in 2 high-grade cancer cell lines showed opposing functional roles. Compared to the non-silencing control, HTB-5 cells, showed decreased cellular proliferation and increased sensitivity to cisplatin, whereas HTB-9 cells, showed increased cell numbers and increased resistance to cisplatin. CONCLUSION: We conclude that our results in bladder cancer are consistent with the conflicting data reported in other cancers, and that SATB1 might have different roles in cancer dependent on genetic background and stage of the cancer.

Optogenetic activation of Plexin-B1 reveals contact repulsion between osteoclasts and osteoblasts.

During bone remodelling, osteoclasts induce chemotaxis of osteoblasts and yet maintain spatial segregation. We show that osteoclasts express the repulsive guidance factor Semaphorin 4D and induce contact inhibition of locomotion (CIL) in osteoblasts through its receptor Plexin-B1. To examine causality and elucidate how localized Plexin-B1 stimulation may spatiotemporally coordinate its downstream targets in guiding cell migration, we develop an optogenetic tool for Plexin-B1 designated optoPlexin. Precise optoPlexin activation at the leading edge of migrating osteoblasts readily induces local retraction and, unexpectedly, distal protrusions to steer cells away. These morphological changes are accompanied by reorganization of Myosin II, PIP3, adhesion and active Cdc42. We attribute the resultant repolarization to RhoA/ROCK-mediated redistribution of ß-Pix, which activates Cdc42 and promotes protrusion. Thus, our data demonstrate a causal role of Plexin-B1 for CIL in osteoblasts and reveals a previously unknown effect of Semaphorin signalling on spatial distribution of an activator of cell migration.

Spontaneous Glutamatergic Synaptic Activity Regulates Constitutive COX-2 Expression in Neurons: OPPOSING ROLES FOR THE TRANSCRIPTION FACTORS CREB (cAMP RESPONSE ELEMENT BINDING) PROTEIN AND Sp1 (STIMULATORY PROTEIN-1).

Burgeoning evidence supports a role for cyclooxygenase metabolites in regulating membrane excitability in various forms of synaptic plasticity. Two cyclooxygenases, COX-1 and COX-2, catalyze the initial step in the metabolism of arachidonic acid to prostaglandins. COX-2 is generally considered inducible, but in glutamatergic neurons in some brain regions, including the cerebral cortex, it is constitutively expressed. However, the transcriptional mechanisms by which this occurs have not been elucidated. Here, we used quantitative PCR and also analyzed reporter gene expression in a mouse line carrying a construct consisting of a portion of the proximal promoter region of the mouse COX-2 gene upstream of luciferase cDNA to characterize COX-2 basal transcriptional regulation in cortical neurons. Extracts from the whole brain and from the cerebral cortex, hippocampus, and olfactory bulbs exhibited high luciferase activity. Moreover, constitutive COX-2 expression and luciferase activity were detected in cortical neurons, but not in cortical astrocytes, cultured from wild-type and transgenic mice, respectively. Constitutive COX-2 expression depended on spontaneous but not evoked excitatory synaptic activity and was shown to be N-methyl-d-aspartate receptor-dependent. Constitutive promoter activity was reduced in neurons transfected with a dominant-negative cAMP response element binding protein (CREB) and was eliminated by mutating the CRE-binding site on the COX-2 promoter. However, mutation of the stimulatory protein-1 (Sp1)-binding site resulted in an N-methyl-d-aspartate receptor-dependent enhancement of COX-2 promoter activity. Basal binding of the transcription factors CREB and Sp1 to the native neuronal COX-2 promoter was confirmed. In toto, our data suggest that spontaneous glutamatergic synaptic activity regulates constitutive neuronal COX-2 expression via Sp1 and CREB protein-dependent transcriptional mechanisms.

Prostaglandin-mediated inhibition of PTH-stimulated ß-catenin signaling in osteoblasts by bone marrow macrophages.

Bone marrow macrophages (BMMs), in the presence of cyclooxygenase-2 (Cox2) produced PGE2, secrete an inhibitory factor in response to Rankl that blocks PTH-stimulated osteoblastic differentiation. This study was to determine if the inhibitory factor also blocks PTH-stimulated Wnt signaling. Primary calvarial osteoblasts (POBs) were co-cultured with conditioned medium (CM) from Rankl-treated wild type (WT) BMMs, which make the inhibitory factor, and Cox2 knockout (KO) BMMs, which do not. PTH induced cAMP production was blocked by WT CM but not by KO CM. In the presence of KO CM, PTH induced phosphorylation at ß-catenin serine sites, ser552 and ser675, previously shown to be phosphorylated by protein kinase A (PKA). Phosphorylation was blocked by WT CM and by H89, a PKA inhibitor. PTH did not increase total ß-catenin. PTH-stimulated transcription factor/lymphoid enhancer-binding factor response element activity in POBs was blocked by WT CM and by serum amyloid A (SAA), the human recombinant analog of murine Saa3, which has recently been shown to be the inhibitory factor. In POBs cultured with Cox2 KO CM, PTH increased expression of multiple genes associated with the anabolic actions of PTH and decreased expression of Wnt antagonists. This differential regulation of gene expression was not seen in POBs cultured with WT CM. These data highlight the ability of PTH to phosphorylate ß-catenin directly via PKA and demonstrate the ability of a Cox2-dependent inhibitory factor, secreted by Rankl-stimulated BMMs, to abrogate PTH stimulated ß-catenin signaling. Our results suggest that PTH can stimulate a novel negative feedback of its anabolic actions by stimulating Rankl and Cox2 expression.

Serum Amyloid A3 Secreted by Preosteoclasts Inhibits Parathyroid Hormone-stimulated cAMP Signaling in Murine Osteoblasts.

Continuous parathyroid hormone (PTH) blocks its own osteogenic actions in marrow stromal cell cultures by inducing Cox2 and receptor activator of nuclear factor κB ligand (RANKL) in the osteoblastic lineage cells, which then cause the hematopoietic lineage cells to secrete an inhibitor of PTH-stimulated osteoblast differentiation. To identify this inhibitor, we used bone marrow macrophages (BMMs) and primary osteoblasts (POBs) from WT and Cox2 knock-out (KO) mice. Conditioned medium (CM) from RANKL-treated WT, but not KO, BMMs blocked PTH-stimulated cAMP production in POBs. Inhibition was reversed by pertussis toxin (PTX), which blocks Gαi/o activation. Saa3 was the most highly differentially expressed gene in a microarray comparison of RANKL-treated WT versus Cox2 KO BMMs, and RANKL induced Saa3 protein secretion only from WT BMMs. CM from RANKL-stimulated BMMs with Saa3 knockdown did not inhibit PTH-stimulated responses in POBs. SAA added to POBs inhibited PTH-stimulated cAMP responses, which was reversed by PTX. Selective agonists and antagonists of formyl peptide receptor 2 (Fpr2) suggested that Fpr2 mediated the inhibitory actions of Saa3 on osteoblasts. In BMMs committed to become osteoclasts by RANKL treatment, Saa3 expression peaked prior to appearance of multinucleated cells. Flow sorting of WT marrow revealed that Saa3 was secreted only from the RANKL-stimulated B220(-) CD3(-)CD11b(-/low) CD115(+) preosteoclast population. We conclude that Saa3 secretion from preosteoclasts, induced by RANKL in a Cox2-dependent manner, inhibits PTH-stimulated cAMP signaling and osteoblast differentiation via Gαi/o signaling. The induction of Saa3 by PTH may explain the suppression of bone formation when PTH is applied continuously and may be a new therapeutic target for osteoporosis.

Cyclooxygenase-2 suppresses the anabolic response to PTH infusion in mice.

We previously reported that the ability of continuously elevated PTH to stimulate osteoblastic differentiation in bone marrow stromal cell cultures was abrogated by an osteoclastic factor secreted in response to cyclooxygenase-2 (Cox2)-produced prostaglandin E2. We now examine the impact of Cox2 (Ptgs2) knockout (KO) on the anabolic response to continuously elevated PTH in vivo. PTH (40 µg/kg/d) or vehicle was infused for 12 or 21 days in 3-mo-old male wild type (WT) and KO mice in the outbred CD-1 background. Changes in bone phenotype were assessed by bone mineral density (BMD), µCT and histomorphometry. PTH infusion for both 12 and 21 days increased femoral BMD in Cox2 KO mice and decreased BMD in WT mice. Femoral and vertebral trabecular bone volume fractions were increased in KO mice, but not in WT mice, by PTH infusion. In the femoral diaphysis, PTH infusion increased cortical area in Cox2 KO, but not WT, femurs. PTH infusion markedly increased trabecular bone formation rate in the femur, serum markers of bone formation, and expression of bone formation-related genes, growth factors, and Wnt target genes in KO mice relative to WT mice, and decreased gene expression of Wnt antagonists only in KO mice. In contrast to the differential effects of PTH on anabolic factors in WT and KO mice, PTH infusion increased serum markers of resorption, expression of resorption-related genes, and the percent bone surface covered by osteoclasts similarly in both WT and KO mice. We conclude that Cox2 inhibits the anabolic, but not the catabolic, effects of continuous PTH. These data suggest that the bone loss with continuously infused PTH in mice is due largely to suppression of bone formation and that this suppression is mediated by Cox2.

Increased expression of L-selectin (CD62L) in high-grade urothelial carcinoma: A potential marker for metastatic disease.

INTRODUCTION: L-Selectin (CD62L) is a vascular adhesion molecule constitutively expressed on leukocytes with a primary function of directing leukocyte migration and homing of lymphocytes to lymph nodes. In a gene expression microarray study comparing laser-captured microdissected high-grade muscle-invasive bladder cancer (MIBC) without prior treatment and low-grade bladder cancer (LGBC) human samples, we found CD62L to be the highest differentially expressed gene. We sought to examine the differential expression of CD62L in MIBCs and its clinical relevance. METHODS: Unfixed fresh and formalin-fixed paraffin-embedded human bladder cancer specimens and serum samples were obtained from the University of Connecticut Health Center tumor bank. Tumor cells were isolated from frozen tumor tissue sections by laser-captured microdissected followed by RNA isolation. Quantitative polymerase chain reaction was used to validate the level of CD62L transcripts. Immunohistochemistry and enzyme-linked immunosorbent assay were performed to evaluate the CD62L protein localization and expression level. Flow cytometry was used to identify the relative number of cells expressing CD62L in fresh tumor tissue. In silico studies were performed using the Oncomine database. RESULTS: Immunostaining showed a uniformly higher expression of CD62L in MIBC specimens vs. LGBCs specimens. Further, CD62L localization was seen in foci of metastatic tumor cells in lymph node specimens from patients with high-grade MIBC and known nodal involvement. Up-regulated expression of CD62L was also observed by flow cytometric analysis of freshly isolated tumor cells from biopsies of high-grade cancers vs. LGBC specimens. Circulating CD62L levels were also found to be higher in serum samples from patients with high-grade metastatic vs. high-grade nonmetastatic MIBC. In addition, in silico analysis of Oncomine Microarray Database showed a significant correlation between CD62L expression and tumor aggressiveness and clinical outcomes. CONCLUSION: These data confirm the expression of CD62L on urothelial carcinoma cells and suggest that CD62L may serve as biomarker to predict the presence of or risk for developing metastatic disease in patients with bladder cancer.

Randomized double-blind placebo-controlled trial of celecoxib for oral mucositis in patients receiving radiation therapy for head and neck cancer.

OBJECTIVES: Oral mucositis (OM) is a painful complication of radiation therapy (RT) for head and neck cancer (H&NC). OM can compromise nutrition, require opioid analgesics and hospitalization for pain control, and lead to treatment interruptions. Based on the role of inflammatory pathways in OM pathogenesis, we investigated effect of cyclooxygenase-2 (COX-2) inhibition on severity and morbidity of OM. METHODS: In this double-blind placebo-controlled trial, 40 H&NC patients were randomized to daily use of 200 mg celecoxib or placebo, for the duration of RT. Clinical OM, normalcy of diet, pain scores, and analgesic use were assessed 2-3 times/week by blinded investigators during the 6-7 week RT period, using validated scales. RESULTS: Twenty subjects were randomized to each arm, which were similar with respect to tumor location, radiation dose, and concomitant chemotherapy. In both arms, mucositis and pain scores increased over course of RT. Intention-to-treat analyses demonstrated no significant difference in mean Oral Mucositis Assessment Scale (OMAS) scores at 5000 cGy (primary endpoint). There was also no difference between the two arms in mean OMAS scores over the period of RT, mean worst pain scores, mean normalcy of diet scores, or mean daily opioid medication use in IV morphine equivalents. There were no adverse events attributed to celecoxib use. CONCLUSIONS: Daily use of a selective COX-2 inhibitor, during period of RT for H&NC, did not reduce the severity of clinical OM, pain, dietary compromise or use of opioid analgesics. These findings also have implications for celecoxib use in H&NC treatment regimens (NCT00698204).

Macrophage migratory inhibitory factor promotes bladder cancer progression via increasing proliferation and angiogenesis.

Macrophage migratory inhibitory factor (MIF) is a proinflammatory cytokine shown to promote tumorigenesis. Using the N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) model of bladder cancer, we previously showed that MIF knockout mice display decreased angiogenesis and invasion compared with wild-type. This study examines the role of MIF in bladder cancer via use of oral inhibitors of MIF. In vitro, high-grade bladder cancer cells were treated with recombinant human MIF +/- (rhMIF+/-) inhibitor. Measurements included cell counts, proliferation by (3)H-thymidine incorporation (TdR), extracellular signal-regulated kinase (ERK) phosphorylation by western blot analysis, messenger RNA (mRNA) expression by quantitative PCR and protein secretion by enzyme-linked immunosorbent assay. Treatment with rhMIF increased ERK phosphorylation, cell counts, TdR and mRNA expression and protein secretion of vascular endothelial growth factor, which were blocked by specific inhibitors of ERK and MIF. In vivo, 3-month-old male C57Bl/6 mice were given BBN for 22 and 16 weeks in study 1 and study 2, respectively. Mice (n = 8-10 per group) were gavaged with vehicle or doses of MIF inhibitors daily from weeks 16-22 in both studies. Average bladder weights, reflecting tumor mass, tumor stage/burden, mitotic rate and proliferation indices, and microvessel densities were reduced in inhibitor groups versus controls. In summary, MIF promotes bladder cancer via increasing cell proliferation and angiogenesis and oral inhibitors of MIF may prove useful in treatment of this disease.

Prostaglandin E2 acts via bone marrow macrophages to block PTH-stimulated osteoblast differentiation in vitro.

Intermittent PTH is the major anabolic therapy for osteoporosis while continuous PTH causes bone loss. PTH acts on the osteoblast (OB) lineage to regulate bone resorption and formation. PTH also induces cyclooxygenase-2 (COX-2), producing prostaglandin E2 (PGE(2)) that can act on both OBs and osteoclasts (OCs). Because intermittent PTH is more anabolic in Cox-2 knockout (KO) than wild type (WT) mice, we hypothesized COX-2 might contribute to the effects of continuous PTH by suppressing PTH-stimulated differentiation of mesenchymal stem cells into OBs. We compared effects of continuous PTH on bone marrow stromal cells (BMSCs) and primary OBs (POBs) from Cox-2 KO mice, mice with deletion of PGE(2) receptors (Ptger(4) and Ptger(2) KO mice), and WT controls. PTH increased OB differentiation in BMSCs only in the absence of COX-2 expression or activity. In the absence of COX-2, PTH stimulated differentiation if added during the first week of culture. In Cox-2 KO BMSCs, PTH-stimulated differentiation was prevented by adding PGE(2) to cultures. Co-culture of POBs with M-CSF-expanded bone marrow macrophages (BMMs) showed that the inhibition of PTH-stimulated OB differentiation required not only COX-2 or PGE(2) but also BMMs. Sufficient PGE(2) to mediate the inhibitory effect was made by either WT POBs or WT BMMs. The inhibitory effect mediated by COX-2/PGE(2) was transferred by conditioned media from RANKL-treated BMMs and could be blocked by osteoprotegerin, which interferes with RANKL binding to its receptor on OC lineage cells. Deletion of Ptger(4), but not Ptger(2), in BMMs prevented the inhibition of PTH-stimulated OB differentiation. As expected, PGE(2) also stimulated OB differentiation, but when given in combination with PTH, the stimulatory effects of both were abrogated. These data suggest that PGE(2), acting via EP4R on BMMs committed to the OC lineage, stimulated secretion of a factor or factors that acted to suppress PTH-stimulated OB differentiation. This suppression of OB differentiation could contribute to the bone loss seen with continuous PTH in vivo.

Basal bone phenotype and increased anabolic responses to intermittent parathyroid hormone in healthy male COX-2 knockout mice.

Cyclooxygenase-2 (COX-2) knockout (KO) mice in inbred strains can have renal dysfunction with secondary hyperparathyroidism (HPTH), making direct effects of COX-2 KO on bone difficult to assess. COX-2 KO mice in an outbred CD-1 background did not have renal dysfunction but still had two-fold elevated PTH compared to wild type (WT) mice. Compared to WT mice, KO mice had increased serum markers of bone turnover, decreased femoral bone mineral density (BMD) and cortical bone thickness, but no differences in trabecular bone volume by microCT or dynamic histomorphometry. Because PTH is a potent inducer of COX-2 and prostaglandin (PG) production, we examined the effects of COX-2 KO on bone responses after 3 weeks of intermittent PTH. Intermittent PTH increased femoral BMD and cortical bone area more in KO mice than in WT mice and increased trabecular bone volume in the distal femur in both WT and KO mice. Although not statistically significant, PTH-stimulated increases in trabecular bone tended to be greater in KO mice than in WT mice. PTH increased serum markers of bone formation and resorption more in KO than in WT mice but increased the ratio of osteoblastic surface-to-osteoclastic surface only in KO mice. PTH also increased femoral mineral apposition rates and bone formation rates in KO mice more than in WT mice. Acute mRNA responses to PTH of genes that might mediate some anabolic and catabolic effects of PTH tended to be greater in KO than WT mice. We conclude that (1) the basal bone phenotype in male COX-2 KO mice might reflect HPTH, COX-2 deficiency or both, and (2) increased responses to intermittent PTH in COX-2 KO mice, despite the presence of chronic HPTH, suggest that absence of COX-2 increased sensitivity to PTH. It is possible that manipulation of endogenous PGs could have important clinical implications for anabolic therapy with PTH.

Prostaglandins in bone: bad cop, good cop?

Prostaglandins (PGs) are multifunctional regulators of bone metabolism that stimulate both bone resorption and formation. PGs have been implicated in bone resorption associated with inflammation and metastatic bone disease, and also in bone formation associated with fracture healing and heterotopic ossification. Recent studies have identified roles for inducible cyclooxygenase (COX)-2 and PGE(2) receptors in these processes. Although the effects of PGs have been most often associated with cAMP production and protein kinase A activation, PGs can engage an extensive G-protein signaling network. Further analysis of COX-2 and PG receptors and their downstream G-protein signaling in bone could provide important clues to the regulation of skeletal cell growth in both health and disease.

Parathyroid hormone induction of cyclooxygenase-2 in murine osteoblasts: role of the calcium-calcineurin-NFAT pathway.

Murine MC3T3-E1 and MC-4 cells were stably transfected with -371/+70 bp of the murine cyclooxygenase-2 (COX-2) promoter fused to a luciferase reporter (Pluc371) or with Pluc371 carrying site-directed mutations. Mutations were made in (1) the cAMP response element (CRE) at -57/-52 bp, (2) the activating protein-1 (AP-1)-binding site at -69/-63 bp, (3) the nuclear factor of activated T-cells (NFAT)-binding site at -77/-73 bp, and (4) both the AP-1 and NFAT sites, which comprise a composite consensus sequence for NFAT/AP-1. Single mutation of CRE, AP-1, or NFAT sites decreased parathyroid hormone (PTH)-stimulated COX-2 promoter activity 40% to 60%, whereas joint mutation of NFAT and AP-1 abrogated the induction. On electrophoretic mobility shift analysis, PTH stimulated binding of phosphorylated CREB to an oligonucleotide spanning the CRE and binding of NFATc1, c-Fos, and c-Jun to an oligonucleotide spanning the NFAT/AP-1 composite site. Mutation of the NFAT site was less effective than mutation of the AP-1 site in competing binding to the composite element, suggesting that cooperative interactions of NFATc1 and AP-1 are more dependent on NFAT than on AP-1. Both PTH and forskolin, an activator of adenylyl cyclase, stimulated NFATc1 nuclear translocation. PTH- and forskolin-stimulated COX-2 promoter activity was inhibited 56% to 80% by calcium chelation or calcineurin inhibitors and 60% to 98% by protein kinase A (PKA) inhibitors. These results indicate an important role for the calcium-calcineurin-NFAT signaling pathway in the PTH induction of COX-2 and suggest that cross-talk between the cAMP/PKA pathway and the calcium-calcineurin-NFAT pathway may play a role in other functions of PTH in osteoblasts.

Role of the cyclooxygenase pathway in chemotherapy-induced oral mucositis: a pilot study.

GOALS: Oral mucositis can be a significant and dose-limiting complication of high-dose cancer therapy. Mucositis is a particularly severe problem in patients receiving myeloablative chemotherapy prior to bone marrow or hematopoetic stem cell transplant (HSCT). The cyclooxygenase (COX) pathway mediates tissue injury and pain through upregulation of pro-inflammatory prostaglandins, including prostaglandin E2 (PGE2) and prostacyclin (PGI2). The objective of this small (n = 3) pilot study was to examine the role of the COX pathway in causing mucosal injury and pain in chemotherapy-induced oral mucositis. MATERIALS AND METHODS: We collected blood, saliva, and oral mucosal biopsy specimens from three autologous HSCT patients at the following time-points before and after administration of conditioning chemotherapy: Day -10, +10, +28, and +100, where day 0 is day of transplant. RNA extracted from full-thickness tissue samples was measured by RT-PCR for the following: COX-1, COX-2, microsomal prostaglandin E synthase (mPGES), IL-1beta, and TNF-alpha. Blood and saliva samples were measured by ELISA for PGE2 and PGI2, which are markers of COX activity. Severity of oral mucositis was determined using the Oral Mucositis Index. Severity of pain due to oral mucositis was measured using a Visual Analog Scale. Relationships between the different variables were examined using Spearman rank correlation coefficients. MAIN RESULTS: Mean mucositis and pain scores increased significantly after administration of chemotherapy and then gradually declined. The correlation between changes in mucositis and pain scores was strong and statistically significant. The following additional correlations were statistically significant: between tissue COX-1 and pain; between tissue mPGES and pain; between salivary PGE1 and pain; between salivary PGI2 and pain. Other relationships were not statistically significant. CONCLUSIONS: Our finding of significant associations of pain scores with tissue COX-1 and mPGES, as well as salivary prostaglandins, is suggestive of a role for the cyclooxygenase pathway in mucositis, possibly via upregulation of pro-inflammatory prostaglandins. However, our small sample size may have contributed to the lack of significant associations between COX-2 and other inflammatory mediators with mucosal injury and pain. Thus, additional studies with larger numbers of subjects are warranted to confirm the involvement of the cyclooxygenase pathway in chemotherapy-induced mucositis.

Bone morphogenetic protein 2 enhances PGE(2)-stimulated osteoclast formation in murine bone marrow cultures.

Bone morphogenetic protein 2 (BMP-2) is used clinically to stimulate bone formation and accelerate fracture repair. Adding prostaglandin (PG) E(2) or PGE(2) receptor agonists to BMP-2 has been proposed to improve BMP-2 efficacy. However, this may enhance bone resorption, since PGE(2) can increase receptor activator of NF-kappaB ligand (RANKL) expression and decrease osteoprotegerin (OPG) expression in osteoblasts, and the RANKL:OPG ratio is critical for osteoclast formation. We used bone marrow (BM) cultures and BM macrophage (BMM) cultures from outbred CD1 mice to examine effects on osteoclast formation of BMP-2 and PGE(2). In BM cultures, which contain both osteoblastic and osteoclastic lineage cells, BMP-2 (100 ng/ml) alone did not increase osteoclast formation but enhanced the peak response to PGE(2) by 1.6-9.6-fold. In BMM cultures, which must be treated with RANKL because they do not contain osteoblastic cells, BMP-2 did not increase osteoclast formation, with or without PGE(2). Our results suggest that BMP-2 can increase osteoclast formation in response to PGE(2) by increasing the RANKL:OPG ratio in osteoblasts, which may have therapeutic implications for the use of BMP-2.