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Randy N Rosier

Publications and source records attributed to Randy N Rosier.

At least 37 records · Page 2Linked to original sources
PubMed2004

CREB Cooperates with BMP-stimulated Smad signaling to enhance transcription of the Smad6 promoter.

Growth plate chondrocytes integrate a multitude of growth factor signals during maturation. PTHrP inhibits maturation through stimulation of PKA/CREB signaling while the bone morphogenetic proteins (BMPs) stimulate maturation through Smad mediated signaling. In this manuscript, we show that interactions between CREB and the BMP associated Smads are promoter specific, and demonstrate for the first time the requirement of CREB signaling for Smad mediated activation of a BMP responsive region of the Smad6 promoter. The 28 base pairs (bp) BMP responsive element of the Smad6 promoter contains an 11 bp Smad binding region and an adjacent 17 bp region in which we characterize a putative CRE site. PKA/CREB gain of function enhanced BMP stimulation of this reporter, while loss of CREB function diminished transcriptional activity. In contrast, ATF-2 and AP-1 transcription factors had minimal effects. Electrophoretic mobility shift assay (EMSA) confirmed CREB binding to the Smad6 promoter element. Mutations eliminating binding resulted in loss of transcriptional activity, while mutations that maintained CREB binding had continued reporter activation by CREB and BMP-2. The Smad6 gene was similarly regulated by CREB. Dominant negative CREB reduced BMP-2 stimulated Smad6 gene transcription by 50%, but markedly increased BMP-2 mediated stimulation of colX and Ihh expression. In contrast, PTHrP which activates CREB signaling, blocked the stimulatory effect of BMP-2 on colX and Ihh, but minimally inhibited the stimulatory effect of BMP on Smad6. These findings are the first to demonstrate a cooperative association between CREB and BMP regulated Smads in cells from vertebrates and demonstrate that promoter-specific rather than generalized interactions between PKA/CREB and BMP signaling regulate gene expression in chondrocytes.

Animals↗
PubMed2004

Primary murine limb bud mesenchymal cells in long-term culture complete chondrocyte differentiation: TGF-beta delays hypertrophy and PGE2 inhibits terminal differentiation.

In vitro models of endochondral bone formation using both primary and immortalized cells have provided insight regarding factors and signaling pathways involved in chondrocyte maturation and endochondral bone formation. However, primary murine cell culture models of chondrocyte differentiation have not been established but have enormous potential due to the possible use of cells from transgenic and knockout animals. Here, we show that stage E11.5 embryonic murine limb bud mesenchymal stem cells in micromass cell culture progress through the stages of chondrogenesis, chondrocyte hypertrophy, terminal differentiation, and matrix calcification. This cell culture system recapitulated the sequential expression of genes that characterize chondrocyte differentiation, including Sox9, col2, colX, MMP13, VEGF, and osteocalcin. TGF-beta treatment for up to 21 days markedly delayed the rate of chondrocyte maturation and inhibited matrix calcification and its related gene expression. In TGF-beta-treated cultures, the hypertrophic and terminal differentiation markers colX, VEGF, MMP13, and osteocalcin were reduced or absent. PGE2 had minimal effects on chondrocyte hypertrophy but delayed terminal differentiation and matrix calcification. Thus, primary murine mesenchymal cells sequentially differentiate through the various stages of chondrocyte maturation and establish a model whereby the role of specific signaling molecules can be examined in cells derived from transgenic or knockout mice.

Animals↗
PubMed2004

PGE2 and IL-6 production by fibroblasts in response to titanium wear debris particles is mediated through a Cox-2 dependent pathway.

Aseptic loosening of orthopaedic implants is precipitated by wear debris-induced osteolysis. Central to this process are the pro-inflammatory mediators that are produced in response to wear by the fibroblastic cells, which comprise the majority of periprosthetic membranes. Since this pro-inflammatory cascade is mediated by a plethora of factors with redundant functions, it is imperative to establish a hierarchy. Two well-known fibroblast derived pro-inflammatory factors that stimulate wear debris-induced osteoclastic resorption are prostaglandin E2 (PGE2) and IL-6. However, their relationship to each other in this process is poorly defined. Here we show immunohistochemistry of retrieval membranes indicating that COX-2 is the principal cyclooxygenase responsible for PGE2 production in fibroblasts around failed implants. We also performed in vitro experiments with fibroblasts derived from wild-type (WT), COX-1 (-/-) and COX-2 (-/-) mice, which demonstrated that COX-2 is required for Ti wear debris-induced PGE2 production. Interestingly, COX-2 was also required for IL-6 production in these assays, which could be rescued by the addition of exogenous PGE2 (10(-6) M). Pharmacology studies that utilized the COX-1 selective inhibitor SC 560, the COX-2 selective inhibitor celecoxib, and the nonselective COX inhibitor indomethacin confirmed these results. Taken together, these results indicate that selective inhibition of prostaglandin signaling could favorably impact aseptic loosening beyond its direct effects on PGE2 synthesis, in that it inhibits downstream pro-inflammatory/pro-osteoclastic cytokine production.

Animals↗
PubMed2004

The association between environmental lead exposure and bone density in children.

Osteoporosis is a decrease in bone mineral density (BMD) that predisposes individuals to fractures. Although an elderly affliction, a predisposition may develop during adolescence if a sufficient peak BMD is not achieved. Rat studies have found that lead exposure is associated with decreased BMD. However, human studies are limited. We hypothesized that the BMD of children with high lead exposure would be lower than the BMD of children with low lead exposure. We collected data on 35 subjects; 16 had low cumulative lead exposure (mean, 6.5 microg/dL), and 19 had high exposure (mean, 23.6 micro g/dL). All were African American; there was no difference between the groups by sex, age, body mass index, socioeconomic status, physical activity, or calcium intake. Significant differences in BMD between low and high cumulative lead exposure were noted in the head (1.589 vs. 1.721 g/cm2), third lumbar vertebra (0.761 vs. 0.819 g/cm2), and fourth lumbar vertebra (0.712 vs. 0.789 g/cm2). Contrary to our hypothesis, subjects with high lead exposure had a significantly higher BMD than did subjects with low lead exposure. This may reflect a true phenomenon because lead exposure has been reported to accelerate bony maturation by inhibiting the effects of parathyroid hormone-related peptide. Accelerated maturation of bone may ultimately result in a lower peak BMD being achieved in young adulthood, thus predisposing to osteoporosis in later life. Future studies need to investigate this proposed model.

Black or African American↗
PubMed2003

Runx2/Cbfa1 stimulation by retinoic acid is potentiated by BMP2 signaling through interaction with Smad1 on the collagen X promoter in chondrocytes.

Chondrocyte differentiation is a fundamental process during endochondral ossification. Several factors regulate maturation via the activity of downstream signaling pathways that target specific transcription factors and regulate chondrocyte-specific genes. In this study, we investigated the mechanisms involved in the regulation of chick lower sternal chondrocyte maturation upon stimulation by retinoic acid (RA) and the bone morphogenetic protein BMP2. RA-induced Runx2 in lower sternal chondrocyte cultures and over-expression of wild-type (WT) Runx2 enhanced colX and alkaline phosphatase activity, while over-expression of dominant negative Runx2 was inhibitory. Furthermore, WT Runx2 enhanced the effects of both BMP2 and RA on colX expression, while the effects of both growth factors were completely blocked in cultures over-expressing dominant negative Runx2. Similarly, WT Runx2 enhanced the induction of colX by Smad1. Smad1 and Runx2 were found to act cooperatively at the chicken type X collagen promoter and elimination of either the putative Smad binding site or Runx2 binding site eliminated responsiveness to BMP2, RA, or either of the transcription factors. Altogether the results show cross talk between the BMP-associated Smads and Runx2 during chondrocyte differentiation and dependence upon both signals for induction of the type X collagen promoter. Factors or signals that alter either of these transcription factors regulate the rate of chondrocyte differentiation.

Animals↗
PubMed2003

ATF-2 cooperates with Smad3 to mediate TGF-beta effects on chondrocyte maturation.

This study demonstrates that ATF-2 cooperates with Smad3 to regulate the rate of chondrocyte maturation in response to TGF-beta. ATF-2 was rapidly phosphorylated in chick embryonic cephalic sternal chondrocytes following treatment with TGF-beta, and the effect was dependent upon p38 kinase activity. Transient transfection of both wild-type ATF-2 or Smad3 activated the TGF-beta-responsive reporter, p3TP-Lux, and synergistic effects were observed with ATF-2 and Smad3 coexpression. The effect of Smad3 and ATF-2 alone and in combination on chondrocyte maturation was examined in cultures simultaneously infected with RCAS viruses expressing different viral envelope proteins. When expressed alone, wild-type ATF-2 or Smad3 both inhibit colX expression and partially mimic the effects of exogenous TGF-beta. However, in combination the effects were additive and similar to the inhibitory effects of TGF-beta on colX expression. Loss of function experiments using dominant negative ATF-2 or Smad3 partially blocked the inhibitory effect of TGF-beta on colX, while together the blockade was complete. Similar effects were observed with another TGF-beta-responsive gene, PTHrP. However, the induction of colX by BMP-2 was not affected by overexpression of either wild-type or dominant negative ATF-2, indicating specificity for TGF-beta signaling. In contrast, although TGF-beta does not activate CRE/CREB signaling, dominant negative CREB enhanced colX expression in control and in TGF-beta and BMP-2-treated cultures. Thus, ATF-2 regulates chondrocyte maturation as a direct target of TGF-beta signaling while CREB regulates differentiation by targeting genes independent of the individual signaling effects of TGF-beta or BMP-2.

Activating Transcription Factor 2↗
PubMed2003

Clinical relevance of increased retinoid and cAMP transcriptional programs in tumor cells rendered non-malignant by dominant negative inhibition of NFkappaB.

We previously reported reciprocal regulation of extracellular matrix degrading enzymes and their endogenous inhibitors by NFkappaB. As such, dominant negative inhibition of NFkappaB in a murine lung alveolar carcinoma cell, Line 1, afforded a decrease in malignant proclivity [Cancer Res. 60(23) (2000) 6557-6562]. Contrasting the gene expression profile of malignant Line 1 tumor cells (WT-Line 1) and their non-malignant counterparts transduced with a dominant negative inhibitor of NFkappaB (mIkappaB-Line 1), we observed upregulated retinoic acid receptors (RARs) and the cAMP response element modulator (CREM), in mIkappaB-Line 1 tumor cells, and utilized heterologous promoter-reporter constructs to confirm enhanced responsiveness. We translate these findings by inducing retinoid and cAMP transcriptional programs in WT-Line 1 tumor cells with pharmacologic doses of all-trans retinoic acid (at-RA) and pentoxyfilline (PTX), respectively, and demonstrate suppression of NFkappaB activity, tumor cell derived matrix metalloprotease 9 activity, tumor cell invasiveness in vitro and spontaneous metastasis in vivo. Our results are consistent with the putative role of retinoids and cAMP in the malignant reversion of tumor cells and illustrative of the binary nature of transcriptional programs that modulate malignant progression.

Animals↗
PubMed2003

Smad6 is induced by BMP-2 and modulates chondrocyte differentiation.

BMPs regulate cartilage differentiation and have been approved for clinical use as stimulators of bone repair. BMP signaling is complex and there are multiple potential points of regulation, including modulation of Smad signaling, which is inhibited by both Smad6 and Smad7. In the current manuscript we assessed the expression and biological function of Smad6 during chondrocyte differentiation. We found that the induction of chondrocyte differentiation by BMP-2 in chicken sternal embryonic chondrocytes was accompanied by a marked increase in Smad6 mRNA and protein levels. A morpholino antisense oligonucleotide complementary to Smad6 reduced the expression of Smad6 protein and enhanced the stimulatory effect of BMP-2 on both colX and alkaline phosphatase activity. In contrast, over-expression of Smad6 blocked BMP-2 mediated induction of the type X collagen promoter, b2-640 Luc. Therefore, expression studies as well as gain and loss of function experiments suggest that Smad6 participates in an important negative feedback loop whereby BMP-2 mediated effects on chondrocyte differentiation are reduced by induction of Smad6. Additional studies are required to determine the extent to which this pathway participates in pathologic processes involving cartilage.

Animals↗
PubMed2003

NFkappaB: a pivotal transcription factor in prostate cancer metastasis to bone.

Prostate cancers frequently metastasize to bone and this accounts for substantial morbidity. We investigated the potential role of the transcription factor NFkappaB as a central regulator of prostate cancer metastasis using the prostate adenocarcinoma cell line, PC-3, in a series of in vitro studies. Wild type PC-3 cells (PC-3.WT) have high basal levels of NFkappaB signaling, otherwise absent in PC-3 cells stably expressing a mutant form of the inhibitory kappa B (IkappaB) protein alpha (PC-3.mIkappaB). Although PC-3.WT cells in co-culture with rat bone marrow cells enhance bone resorption, no increase was observed in co-cultures with PC-3.mIkappaB cells. Similarly, although PC-3.WT cells were invasive in a chicken chorioallantoic membrane extravasation model, PC-3.mIkappaB cells lose this capacity to invade. NFkappaB reciprocally regulated genes involved in cellular invasion, with upregulation of MMP-9 and downregulation of its inhibitor, TIMP-1 in PC-3.WT cells, whereas MMP-9 was downregulated and TIMP-1 was upregulated in PC-3.mIkappaB cells. Finally, high basal gene and protein expression of the osteoclast-activating cytokine IL-6, observed in PC-3.WT cells, was absent in PC-3.mIkappaB cells. These in vitro experiments suggest NFkappaB as an important target to prevent prostate cancer bone metastasis and provide a rationale for further study of this transcription factor in metastatic disease.

Adenocarcinoma↗
PubMed2003

The mevalonate synthesis pathway as a therapeutic target in cancer.

Farnesyl transferase inhibitors have emerged as bona fide anticancer agents whereas the development of geranylgeranyl transferase inhibitors has been mitigated by overt systemic toxicities. Evidence suggests that the therapeutic value of farnesyl transferase inhibitors is an indirect result of perturbations in the function of geranylgeranylated Rho proteins. To address this question, we used inhibitors of the mevalonate synthesis pathway to decrease cellular levels of farnesly and geranylgeranly isoprenoids and supplemented our culture systems with exogenous isoprenoids accordingly. Using a murine lung alveolar carcinoma cell line (Line 1), we report a dose-dependent inhibition of tumor cell proliferation, adhesion and invasiveness, in response to alendronate (3-30 micromol/L) and mevastatin (1-10 micromol/L). Supplementation of cultures with geranylgeranyl pyrophosphates (100 micromol/L) was observed to rescue drug-induced phenotypic changes whereas farnesyl pyrophosphate (100 micromol/L) had a minimal effect. Our observations highlight the mevalonate synthesis pathway as a target for anticancer therapies and suggest a greater role for geranylgeranylated proteins in cellular processes germane to cancer.

Acute-Phase Proteins↗
PubMed2003

Retinoic acid stimulates chondrocyte differentiation and enhances bone morphogenetic protein effects through induction of Smad1 and Smad5.

Whereas bone morphogenetic protein (BMP)-signaling events induce maturational characteristics in vitro, recent evidence suggests that the effects of other regulators might be mediated through BMP-signaling events. The present study examines the mechanism through which retinoic acid (RA) stimulates differentiation in chicken embryonic caudal sternal chondrocyte cultures. Both RA and BMP-2 induced expression of the chondrocyte maturational marker, colX, in chondrocyte cultures by 8 d. Though the RA effect was small, it synergistically enhanced the effect of BMP-2 on colX and phosphatase activity. Inhibition of either RA or BMP signaling, with selective inhibitors, interfered with the inductive effects of these agents but also inhibited the complementary pathway, demonstrating a codependence of RA and BMP signaling during chondrocyte maturation. BMP-2 did not enhance the effects of RA on an RA-responsive reporter construct, but RA enhanced basal activity and synergistically enhanced BMP-2 stimulation of the BMP-responsive chicken type X collagen reporter. A similar synergistic interaction between RA and BMP-2 was observed on colX expression. RA did not increase the expression of the type IA BMP receptor but did markedly up-regulate the expression of Smad1 and Smad5 proteins, important participants in the BMP pathway. Inhibition of RA signaling, with the selective inhibitor AGN 193109, blocked RA-mediated induction of the Smad proteins and chondrocyte differentiation. These findings demonstrate that RA induces the expression of BMP-signaling molecules and enhances BMP effects in chondrocytes.

Alkaline Phosphatase↗
PubMed2003

ALK2 functions as a BMP type I receptor and induces Indian hedgehog in chondrocytes during skeletal development.

UNLABELLED: Growth plate chondrocytes integrate multiple signals during normal development. The type I BMP receptor ALK2 is expressed in cartilage and expression of constitutively active (CA) ALK2 and other activated type I BMP receptors results in maturation-independent expression of Ihh in chondrocytes in vitro and in vivo. The findings suggest that BMP signaling modulates the Ihh/PTHrP signaling pathway that regulates the rate of chondrocyte differentiation. INTRODUCTION: Bone morphogenetic proteins (BMPs) have an important role in vertebrate limb development. The expression of the BMP type I receptors BMPR-IA (ALK3) and BMPR-IB (ALK6) have been more completely characterized in skeletal development than ALK2. METHODS: ALK2 expression was examined in vitro in isolated chick chondrocytes and osteoblasts and in vivo in the developing chick limb bud. The effect of overexpression of CA ALK2 and the other type I BMP receptors on the expression of genes involved in chondrocyte maturation was determined. RESULTS: ALK2 was expressed in isolated chick osteoblasts and chondrocytes and specifically mediated BMP signaling. In the developing chick limb bud, ALK2 was highly expressed in mesenchymal soft tissues. In skeletal elements, expression was higher in less mature chondrocytes than in chondrocytes undergoing terminal differentiation. CA ALK2 misexpression in vitro enhanced chondrocyte maturation and induced Ihh. Surprisingly, although parathyroid hormone-related peptide (PTHrP) strongly inhibited CA ALK2 mediated chondrocyte differentiation, Ihh expression was minimally decreased. CA ALK2 viral infection in stage 19-23 limbs resulted in cartilage expansion with joint fusion. Enhanced periarticular expression of PTHrP and delayed maturation of the cartilage elements were observed. In the cartilage element, CA ALK2 misexpression precisely colocalized with the expression with Ihh. These findings were most evident in partially infected limbs where normal morphology was maintained. In contrast, BMP-6 had a normal pattern of differentiation-related expression. CA BMPR-IA and CA BMPR-IB overexpression similarly induced Ihh and PTHrP. CONCLUSIONS: The findings show that BMP signaling induces Ihh. Although the colocalization of the activated type I receptors and Ihh suggests a direct BMP-mediated signaling event, other indirect mechanisms may also be involved. Thus, while BMPs act directly on chondrocytes to induce maturation, this effect is counterbalanced in vivo by induction of the Ihh/PTHrP signaling loop. The findings suggest that BMPs are integrated into the Ihh/PTHrP signaling loop and that a fine balance of BMP signaling is essential for normal chondrocyte maturation and skeletal development.

Activin Receptors, Type I↗
PubMed2002

A phage display technique identifies a novel regulator of cell differentiation.

The formation of new bone during the process of bone remodeling occurs almost exclusively at sites of prior bone resorption. In an attempt to discover what regulatory pathways are utilized by osteoblasts to effect this site-specific formation event we probed components of an active bone resorption surface with an osteoblast phage expression library. In these experiments primary cultures of rat osteoblasts were used to construct a phage display library in T7 phage. Tartrate-resistant acid phosphatase (type V) (TRAP) was used as the bait in a biopanning procedure. 40 phage clones with very high affinity for TRAP were sequenced, and of the clones with multiple consensus sequences we identified a regulatory protein that modulates osteoblast differentiation. This protein is the TGFbeta receptor-interacting protein (TRIP-1). Our data demonstrate that TRAP activation of TRIP-1 evokes a TGFbeta-like differentiation process. Specifically, TRIP-1 activation increases the activity and expression of osteoblast alkaline phosphatase, osteoprotegerin, collagen, and Runx2. Moreover, we show that TRAP interacts with TRIP intracellularly, that activation of the TGFbeta type II receptor by TRIP-1 occurs in the presence of TRAP and that the differentiation process is mediated through the Smad2/3 pathway. A final experiment demonstrates that osteoblasts, when cultured in osteoclast lacunae containing TRAP, rapidly and specifically differentiate into a mature bone-forming phenotype. We hypothesize that binding to TRAP may be one mechanism by which the full osteoblast phenotype is expressed during the process of bone remodeling.

Acid Phosphatase↗
PubMed2002

Malignant reversion of a human osteosarcoma cell line, Saos-2, by inhibition of NFkappaB.

Beyond a pivotal role in neoplastic transformation and malignant progression, NFkappaB is intricately involved in bone biology, pointed up by the osteopetrotic phenotype of NFkappaB (p50-p52) double knock-out mice. Osteopetrosis results from intrinsic defects in osteoclastogenesis, loss of osteoclast bone resorptive activity and, questionably, increased osteoblast activity (bone matrix apposition and mineralization). We here report that inhibition of NFkappaB signaling activity in Saos-2 cells results in a marked decrease in cellular proliferation, assessed by the incorporation of radioactive thymidine into cellular DNA. Decreased cellular proliferation was accompanied by the induction of bone morphogenic proteins (BMP) 4, 7, and the osteoblast specific transciption factor, Cbfa1, heralding osteoblast differentiation, given the induction of alkaline phosphatase, osteopontin, and osteocalcin message levels and the attendant increase in matrix deposition and mineralization in vitro. These results point to the negative regulation of osteoblast differentiation by NFkappaB, with implications in the pathogenesis and progression of osteosarcomas.

Alkaline Phosphatase↗
PubMed2002

Growth plate chondrocyte maturation is regulated by basal intracellular calcium.

Among the cellular events that are associated with the process of endochondral ossification is an incremental increase in chondrocyte basal intracellular free Ca(2+) concentration ([Ca(2+)](i)) from 50 to 100 nM. To determine if this rise in [Ca(2+)](i) functionally participates in the maturational process of growth plate chondrocytes (GPCs), we examined its effect on several markers of hypertrophy, including annexin V, bone morphogenetic protein-6, type X collagen, and indian hedgehog. Expression of these genes was determined under conditions either where the Ca(2+) chelator EGTA was used to deplete extracellular Ca(2+) and lower [Ca(2+)](i) to < 50 nM or where the extracellular addition of 5 mM CaCl(2) was used to elevate [Ca(2+)](i) to > 100 nM. Although no effect on the expression of these genes was observed following treatment with 5 mM CaCl(2), 4 mM EGTA significantly inhibited their expression. This effect was recapitulated in sternal chondrocytes and was reversed following withdrawal of EGTA. Based on these findings, we hypothesized that the EGTA-induced suppression of these genes was mediated by a factor whose expression is responsive to changes in basal [Ca(2+)](i). Since EGTA mimicked the effect of parathyroid hormone-related peptide (PTHrP) on GPC maturation, we examined the effect of low [Ca(2+)](i) on PTHrP expression. Suggesting that low [Ca(2+)](i) suppression of hypertrophy was PTHrP-dependent in GPCs, (a) treatment with 4 mM EGTA increased PTHrP expression, (b) the EGTA effect was rescued by blocking PTHrP binding to its receptor with the competitive antagonist TIP(7-39), and (c) EGTA could mimic the PTHrP stimulation of AP-1 binding to DNA. Additionally, PTHrP promoter analysis identified a domain (-1498 to -862, relative to the start codon) involved with conferring Ca(2+) sensitivity to the PTHrP gene. These findings underscore the importance of cellular Ca(2+) in GPC function and suggest that PTHrP action in the growth plate is at least partially regulated by changes in basal [Ca(2+)](i).

Animals↗
PubMed2002

Viral interleukin-10 gene inhibition of inflammation, osteoclastogenesis, and bone resorption in response to titanium particles.

OBJECTIVE: To evaluate the potential of viral interleukin-10 (vIL-10) gene therapy as an approach to prevent wear debris-induced inflammation, osteoclastogenesis, and bone resorption as it relates to periprosthetic osteolysis in patients with total joint replacements. METHODS: Replication-defective adenovirus vectors expressing vIL-10 (AdvIL-10) or LacZ (AdLacZ) target genes were used to transduce fibroblast-like synoviocytes (FLS) in vitro, and the effects of these cells on wear debris-induced proinflammatory cytokine production and receptor activator of nuclear factor kappaB ligand + macrophage colony-stimulating factor splenocyte osteoclastogenesis were assessed by enzyme-linked immunosorbent assay and tartrate-resistant acid phosphatase assay. The effects of AdvIL-10 administration on wear debris-induced osteolysis in vivo were analyzed using the mouse calvaria model, in which AdLacZ was used as the control. RESULTS: In the presence of AdLacZ-infected FLS, titanium particle-stimulated macrophages exhibited a marked increase in secretion of tumor necrosis factor alpha (TNFalpha) (6.5-fold), IL-6 (13-fold), and IL-1 (5-fold). Coculture with AdvIL-10-transduced FLS suppressed cytokine secretion to basal levels, while addition of an anti-IL-10 neutralizing antibody completely blocked this effect. The vIL-10-transduced FLS also inhibited osteoclastogenesis 10-fold in an anti-IL-10-sensitive manner. In vivo, titanium implantation resulted in a 2-fold increase in osteoclasts (P < 0.05) and in a 2-fold increase in sagittal suture area (P < 0.05). This increase over control levels was completely blocked in mice receiving intraperitoneal injections of AdvIL-10, all of whom had measurable serum vIL-10 levels for the duration of the experiment. Immunohistochemistry demonstrated reduced cyclooxygenase 2 and TNFalpha expression in AdvIL-10-infected animals. CONCLUSION: This study demonstrates that gene delivery of vIL-10 inhibits 3 processes critically involved in periprosthetic osteolysis: 1) wear debris-induced proinflammatory cytokine production, 2) osteoclastogenesis, and 3) osteolysis.

Adenoviridae↗
PubMed2002

Immunohistochemical study of receptor activator of nuclear factor kappa-B ligand (RANK-L) in human osteolytic bone tumors.

BACKGROUND AND OBJECTIVES: Osteolytic bone tumors produce intercellular signaling proteins that regulate bone remodeling by altering the rates of osteoclast and osteoblast differentiation and activity. This report examines osteolytic bone tumor expression of receptor activator of nuclear factor B-ligand (RANK-L), a cytokine that is arguably the most critical regulator of osteoclast differentiation and activation. METHODS: This prospective immunohistochemical study examined RANK-L expression in frozen tissues from sixteen surgical specimens of patients who underwent surgery for the treatment of osteolytic bone tumors between 1999 and 2000. RESULTS: RANK-L was positive in 13 of the 16 cases. Primary benign bone tumors, primary malignant bone tumors, and metastasis to bone were positive for RANK-L. CONCLUSIONS: The cells in some, but not all, osteolytic tumors produce the cytokine RANK-L. Further study is necessary to determine in which specific tumors RANK-L is the cytokine responsible for increased osteoclastic activity, and to develop possible therapeutic use of RANK-L antagonists such as osteoprotegerin (OPG).

Bone Neoplasms↗