Olga Voznesensky

Bone Morphogenetic Protein 2 Induces Cyclo-oxygenase 2 in Osteoblasts via a Cbfa1 Binding Site: Role in Effects of Bone Morphogenetic Protein 2 In Vitro and In Vivo

We tested the hypothesis that induction of cyclo‐oxygenase (COX) 2 mediates some effects of bone morphogenetic protein (BMP) 2 on bone. BMP‐2 induced COX‐2 mRNA and prostaglandin (PG) production in cultured osteoblasts. BMP‐2 increased luciferase activity in calvarial osteoblasts from mice transgenic for a COX‐2 promoter‐luciferase reporter construct (Pluc) and in MC3T3‐E1 cells transfected with Pluc. Deletion analysis identified the −300/−213‐bp region of the COX‐2 promoter as necessary for BMP‐2 stimulation of luciferase activity. Mutation of core‐binding factor activity 1 (muCbfa1) consensus sequence (5′‐AACCACA‐3′) at −267/−261 bp decreased BMP‐2 stimulation of luciferase activity by 82%. Binding of nuclear proteins to an oligonucleotide spanning the Cbfa1 site was inhibited or supershifted by specific antibodies to Cbfa1. In cultured osteoblasts from calvariae of COX‐2 knockout (−/−) and wild‐type (+/+) mice, the absence of COX‐2 expression reduced the BMP‐2 stimulation of both ALP activity and osteocalcin mRNA expression. In cultured marrow cells flushed from long bones, BMP‐2 induced osteoclast formation in cells from COX‐2+/+ mice but not in cells from COX‐2−/− mice. In vivo, BMP‐2 (10 μg/pellet) induced mineralization in pellets of lyophilized collagen implanted in the flanks of mice. Mineralization of pellets, measured by microcomputed tomography (μCT), was decreased by 78% in COX‐2−/− mice compared with COX‐2+/+ mice. We conclude that BMP‐2 transcriptionally induces COX‐2 in osteoblasts via a Cbfa1 binding site and that the BMP‐2 induction of COX‐2 can contribute to effects of BMP‐2 on osteoblastic differentiation and osteoclast formation in vitro and to the BMP‐2 stimulation of ectopic bone formation in vivo.

Overexpression of COX-2 in human osteosarcoma cells decreases proliferation and increases apoptosis.

Overexpression of cyclooxygenase-2 (COX-2) is generally considered to promote tumorigenesis. To investigate a potential role of COX-2 in osteosarcoma, we overexpressed COX-2 in human osteosarcoma cells. Saos-2 cells deficient in COX-2 expression were retrovirally transduced or stably transfected with murine COX-2 cDNA. Functional expression of COX-2 was confirmed by Northern and Western analyses and prostaglandin production. Overexpression of COX-2 reduced cell numbers by 50% to 70% compared with controls. Decreased proliferation in COX-2-overexpressing cells was associated with cell cycle prolongation in G(2)-M. Apoptosis, measured by both Annexin V binding assay and terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling staining, was increased in cells overexpressing COX-2, and the increase was not reversed by treatment with NS-398, indicating that the effects were not mediated by prostaglandins. Retroviral COX-2 overexpression in two other human osteosarcoma cell lines, U2OS and TE85, also decreased cell viability. However, in the human colon carcinoma HCT-116 cell line, which is deficient in COX-2, retroviral overexpression of COX-2, at similar efficiency as in Saos-2 cells, increased resistance to apoptosis. Reactive oxygen species (ROS), measured by flow cytometry, were increased by COX-2 overexpression in Saos-2 cells but not in HCT-116 cells. Inhibition of peroxidase activity, but not of COX activity, blocked the ROS increase. Antioxidants blocked the increase in ROS and the increase in apoptosis due to COX-2 overexpression in Saos-2 cells. Our results suggest that (a) COX-2 overexpression in osteosarcoma cells may increase resistance to tumorigenesis by increasing ROS to levels that decrease cell viability and (b) the effects of COX-2 overexpression are cell type/tissue dependent.

Null mutation for Macrophage Migration Inhibitory Factor (MIF) is associated with less aggressive bladder cancer in mice

BackgroundInflammatory cytokines may promote tumorigenesis. Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine with regulatory properties over tumor suppressor proteins involved in bladder cancer. We studied the development of bladder cancer in wild type (WT) and MIF knockout (KO) mice given N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN), a known carcinogen, to determine the role of MIF in bladder cancer initiation and progression.Methods5-month old male C57Bl/6 MIF WT and KO mice were treated with and without BBN. Animals were sacrificed at intervals up to 23 weeks of treatment. Bladder tumor stage and grade were evaluated by H&E. Immunohistochemical (IHC) analysis was performed for MIF and platelet/endothelial cell adhesion molecule 1 (PECAM-1), a measure of vascularization. MIF mRNA was analyzed by quantitative real-time polymerase chain reaction.ResultsPoorly differentiated carcinoma developed in all BBN treated mice by week 20. MIF WT animals developed T2 disease, while KO animals developed only T1 disease. MIF IHC revealed predominantly urothelial cytoplasmic staining in the WT control animals and a shift toward nuclear staining in WT BBN treated animals. MIF mRNA levels were 3-fold higher in BBN treated animals relative to controls when invasive cancer was present. PECAM-1 staining revealed significantly more stromal vessels in the tumors in WT animals when compared to KOs.ConclusionMuscle invasive bladder cancer with increased stromal vascularity was associated with increased MIF mRNA levels and nuclear redistribution. Consistently lower stage tumors were seen in MIF KO compared to WT mice. These data suggest that MIF may play a role in the progression to invasive bladder cancer.

Identification of multiple cis-acting elements mediating the induction of prostaglandin G/H synthase-2 by phorbol ester in murine osteoblastic cells.

The tumor promoter phorbol 13‐myristate 12‐acetate (PMA), the best characterized protein kinase C agonist, frequently regulates gene expression via activation of Fos/Jun (AP‐1) complexes. PMA rapidly and transiently induces prostaglandin G/H synthase‐2 (PGHS‐2) expression in murine osteoblastic MC3T3‐E1 cells, but no functional AP‐1 binding motifs in the 5′‐flanking region have been identified. In MC3T3‐E1 cells transfected with −371/+70 bp of the PGHS‐2 gene fused to a luciferase reporter gene (Pluc), PMA stimulates luciferase activity up to eightfold. Computer analysis of the sequence of the PGHS‐2 promoter region identified three potential AP‐1 elements in the −371/+70 bp region, and deletion analysis suggested that the sequence 5′‐aGAGTCA‐3′ at −69/−63 bp was most likely to mediate stimulation by PMA. Mutation of the putative AP‐1 sequence reduces the ability of PMA to stimulate Pluc activity by 65%. On electrophoretic mobility shift analysis (EMSA), PMA induces binding to a PGHS‐2 probe spanning this sequence, binding is blocked by an unlabeled AP‐1 canonical sequence, and antibodies specific for c‐Jun and c‐Fos inhibit binding. Mutation of this AP‐1 site also causes a small (22%) but significant reduction in the serum stimulation of Pluc activity in transiently transfected MC3T3‐E1 cells. On EMSA, serum induces binding to a PGHS‐2 probe spanning the AP‐1 site, binding is blocked by an unlabeled AP‐1 canonical sequence, and antibodies specific for c‐Jun and c‐Fos inhibit binding. Joint mutation of this AP‐1 site and the nearby CRE site at −56/−52 bp, previously shown to mediate serum, v‐src and PDGF induction of PGHS‐2 in NIH‐3T3 cells, blocks both PMA and serum induction of Pluc activity in MC3T3‐E1 cells. Hence, the AP‐1 and CRE binding sites are jointly but differentially involved in both the PMA and serum stimulation of PGHS‐2 promoter activity. J. Cell. Biochem. 78:197–209, 2000.

Fluid flow induces Rankl expression in primary murine calvarial osteoblasts.

Mechanical loading of bone generates fluid flow within the mineralized matrix that exerts fluid shear stress (FSS) on cells. We examined effects of FSS on receptor activator of nuclear factor κ B ligand (RANKL), a critical factor for osteoclast formation. Primary murine osteoblasts were subjected to pulsatile FSS (5 Hz, 10 dynes/cm2) for 1 h and then returned to static culture for varying times (post‐FSS). Protein levels were measured by Western analysis and mRNA by Northern analysis, RT‐PCR and quantitative PCR. There were 20‐ to 40‐fold increases in RANKL mRNA at 2–4 h post‐FSS. RANKL protein was induced by 2 h post‐FSS and remained elevated for at least 8 h. Effects were independent of cyclooxygenase‐2 activity. Small increases (up to three‐fold) in mRNA of the decoy receptor for RANKL, osteoprotegerin, were seen. Five min of FSS, followed by static culture, was as effective in stimulating RANKL mRNA as 4 h of continuous FSS. FSS induced cAMP activity, and H‐89, a protein kinase A (PKA) inhibitor, blocked the FSS induction of RANKL. H‐89 also inhibited the PKC pathway, but specific PKC inhibitors, GF109203X and Go6983, did not inhibit FSS‐induced RANKL. FSS induced phosphorylation of ERK1/2, and PD98059, an inhibitor of the ERK pathway, inhibited the FSS induction of RANKL mRNA 60%–90%. Thus, brief exposure to FSS resulted in sustained induction of RANKL expression after stopping FSS, and this induction was dependent on PKA and ERK signaling pathways. Increased RANKL after mechanical loading may play a role in initiating bone remodeling. J. Cell. Biochem. 98: 1271–1283, 2006.

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.

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.

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.

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 (PGE2) 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 PGE2 receptors (Ptger4 and Ptger2 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 PGE2 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 PGE2 but also BMMs. Sufficient PGE2 to mediate the inhibitory effect was made by either WT POBs or WT BMMs. The inhibitory effect mediated by COX-2/PGE2 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 Ptger4, but not Ptger2, in BMMs prevented the inhibition of PTH-stimulated OB differentiation. As expected, PGE2 also stimulated OB differentiation, but when given in combination with PTH, the stimulatory effects of both were abrogated. These data suggest that PGE2, 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.

Regulation of the prostaglandin pathway during development of invasive bladder cancer in mice

Prostaglandin E(2) (PGE(2)) is reported to play an important role in tumor development. We explored the differential expression of genes governing production of, and response to, PGE(2) during development of invasive bladder cancer. N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) or vehicle-treated mice (n=4-5) were euthanized after 4-8 weeks (period 1, P1), 12-16 weeks (P2), and 20-23 weeks (P3). Half of each bladder was analyzed histologically and the other half extracted for mRNA analysis by quantitative real-time PCR. Bladders from BBN-treated mice showed progression from submucosal inflammation (P1) to squamous metaplasia/focal CIS (P2) to poorly differentiated, invasive cancer (P3). mRNA levels for the inducible cyclooxygenase, COX-2, were elevated three to fourfold at all time points in BBN-treated mice compared to controls. In contrast, mRNA levels for constitutive COX-1 and cytosolic phospholipase A(2) (cPLA(2)), which releases substrate for COX, were either unchanged or decreased in BBN-treated mice relative to controls. Downstream of COX, mRNA levels of membrane-bound PGE(2) synthase (mPGES-1) were increased 1.7-fold at P1 in BBN bladders but returned to control levels at P2 and P3. mRNA levels for 15-prostaglandin dehydrogenase (PGDH), which inactivates PGE(2), were reduced 50-80% in BBN-treated bladders at all time points. mRNA levels for EP2R and EP4R, receptors for PGE(2), were two to threefold increased at P1, but returned to control levels or below at P3. Hence, increased COX-2 and decreased PDGH expression occurred throughout tumor development, while mPGES-1, EP2R and EP4R were elevated only before development of invasive cancer. We compared expression of these genes in the malignant human urothelial cell lines, HTB-5 and HT-1376, with expression in a benign urothelial cell line, UROtsa. Neither malignant cell line reproduced the complete in vivo pattern, relative to benign cells, but each showed abnormal basal expression of several of the genes downstream of COX-2, but not COX-2 itself. We conclude that components involved in PGE(2) synthesis and activity are differentially regulated during bladder tumor development and the therapeutic efficacy of targeting the various components may vary with stage of tumor development.