FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2060482009> ?p ?o ?g. }

• W2060482009 endingPage "20672" @default.
• W2060482009 startingPage "20659" @default.
• W2060482009 abstract "The receptor activator of NF-κB (RANKL) is the essential signal required for full osteoclast (OC) development, activation, and survival. RANKL is highly expressed in areas of trabecular bone remodeling and inflammatory bone loss, is increased on marrow stromal cells or osteoblasts by osteotropic hormones or cytokines, and is neutralized by osteoprotegerin (OPG), a soluble decoy receptor also crucial for preventing arterial calcification. Vascular endothelial cells (VEC) are critically involved in bone development and remodeling and influence OC recruitment, formation, and activity. Although OCs develop and function in close association with bone VEC and sinusoids, signals mediating their interactions are not well known. Here, we show for the first time that human microvascular endothelial cells (HMVEC) express transcripts for both RANKL and OPG; inflammatory cytokines tumor necrosis factor-α and interleukin-1α elevate RANKL and OPG expression 5–40-fold in HMVEC (with an early OPG peak that declines as RANKL rises), and RANKL protein increases on the surface of tumor necrosis factor-α-activated HMVEC. Cytokine-activated HMVEC promoted the formation, fusion, and bone resorption of OCs formed in co-cultures with circulating human monocytic precursors via a RANKL-mediated mechanism fully antagonized by exogenous OPG. Furthermore, paraffin sections of human osteoporotic fractured bone exhibited increased RANKL immunostaining in vivo on VEC located near resorbing OCs in regions undergoing active bone turnover. Therefore, cytokine-activated VEC may contribute to inflammatory-mediated bone loss via regulated production of RANKL and OPG. VEC-derived OPG may also serve as an autocrine signal to inhibit blood vessel calcification. The receptor activator of NF-κB (RANKL) is the essential signal required for full osteoclast (OC) development, activation, and survival. RANKL is highly expressed in areas of trabecular bone remodeling and inflammatory bone loss, is increased on marrow stromal cells or osteoblasts by osteotropic hormones or cytokines, and is neutralized by osteoprotegerin (OPG), a soluble decoy receptor also crucial for preventing arterial calcification. Vascular endothelial cells (VEC) are critically involved in bone development and remodeling and influence OC recruitment, formation, and activity. Although OCs develop and function in close association with bone VEC and sinusoids, signals mediating their interactions are not well known. Here, we show for the first time that human microvascular endothelial cells (HMVEC) express transcripts for both RANKL and OPG; inflammatory cytokines tumor necrosis factor-α and interleukin-1α elevate RANKL and OPG expression 5–40-fold in HMVEC (with an early OPG peak that declines as RANKL rises), and RANKL protein increases on the surface of tumor necrosis factor-α-activated HMVEC. Cytokine-activated HMVEC promoted the formation, fusion, and bone resorption of OCs formed in co-cultures with circulating human monocytic precursors via a RANKL-mediated mechanism fully antagonized by exogenous OPG. Furthermore, paraffin sections of human osteoporotic fractured bone exhibited increased RANKL immunostaining in vivo on VEC located near resorbing OCs in regions undergoing active bone turnover. Therefore, cytokine-activated VEC may contribute to inflammatory-mediated bone loss via regulated production of RANKL and OPG. VEC-derived OPG may also serve as an autocrine signal to inhibit blood vessel calcification. receptor activator of NF-κB tumor necrosis factor-α TNF-related activation induced cytokine osteoprotegerin ligand osteoprotegerin receptor activator of NF-κB interleukin-1α macrophage colony-stimulating factor 1,25-dihydroxyvitamin D3 dexamethasone parathyroid hormone osteoclast vascular endothelial cells human microvascular endothelial cells (human) osteoblast (human) bone marrow stromal cells peripheral blood mononuclear cell multinucleated cell essential growth medium-microvascular essential basal medium α-minimal essential medium fetal bovine serum Hanks' balanced salt solution paraformaldehyde bovine serum albumin monoclonal antibody polyclonal antibody tartrate resistant acid phosphatase reverse transcriptase-polymerase chain reaction glyceraldehyde-3-phosphate dehydrogenase base pair 4,6-diamidino-2-phenylindole The receptor activator of NF-κB ligand (RANKL),1 also known as osteoprotegerin ligand (OPGL), osteoclast differentiation factor, or TNF-related activation-induced cytokine (TRANCE), is a recently discovered transmembrane molecule of the tumor necrosis factor (TNF) ligand superfamily that is highly expressed in lymphoid tissues and trabecular bone, particularly in areas associated with active bone remodeling or inflammatory osteolysis (1Hofbauer L. Khosla S. Dunstan C. Lacey D. Boyle W. Riggs B. J. Bone Miner. Res. 2000; 15: 2-12Crossref PubMed Scopus (1032) Google Scholar, 2Filvaroff E. Derynck R. Curr. Biol. 1998; 8: R679-R682Abstract Full Text Full Text PDF PubMed Google Scholar, 3Suda T. Takahashi N. Udagawa N. Jimi E. Gillespie M. Martin T. Endocr. Rev. 1999; 20: 345-357Crossref PubMed Google Scholar, 4Wong B. Josien R. Choi Y. J. Leukocyte Biol. 1999; 65: 715-724Crossref PubMed Scopus (199) Google Scholar). RANKL is the essential and final common signal required both in vitro and in vivo for full osteoclastic (OC) differentiation from multipotential hematopoietic precursor cells into mature multinucleated bone-resorptive OCs in the presence of the permissive factor macrophage colony-stimulating factor (M-CSF) (1Hofbauer L. Khosla S. Dunstan C. Lacey D. Boyle W. Riggs B. J. Bone Miner. Res. 2000; 15: 2-12Crossref PubMed Scopus (1032) Google Scholar, 2Filvaroff E. Derynck R. Curr. Biol. 1998; 8: R679-R682Abstract Full Text Full Text PDF PubMed Google Scholar, 3Suda T. Takahashi N. Udagawa N. Jimi E. Gillespie M. Martin T. Endocr. Rev. 1999; 20: 345-357Crossref PubMed Google Scholar, 4Wong B. Josien R. Choi Y. J. Leukocyte Biol. 1999; 65: 715-724Crossref PubMed Scopus (199) Google Scholar, 5Lacey D. Timms E. Tan H.-L. Kelley M. Dunstan C. Burgess T. Elliott R. Colombero A. Elliott G. Scully S. Hsu H. Sullivan J. Hawkins N. Davy E. Capparelli C. Eli A. Qian Y.-X. Kaufman S. Sarosi I. Shalhoub V. Senaldi G. Guo J. Delaney J. Boyle W. Cell. 1998; 93: 165-176Abstract Full Text Full Text PDF PubMed Scopus (4656) Google Scholar, 6Yasuda H. Shima N. Nakagawa N. Yamaguchi K. Kinosaki M. Mochizuki S.-I. Tomoyasu A. Yano K. Goto M. Murakami A. Takahashi N. Suda T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3597-3602Crossref PubMed Scopus (3592) Google Scholar, 7Matsuzaki K. Udagawa N. Takahasi N. Yamaguchi K. Yasuda H. Shima N. Morinaga T. Toyama Y. Yabe Y. Higashio K. Suda T. Biochem. Biophys. Res. Commun. 1998; 246: 199-204Crossref PubMed Scopus (322) Google Scholar). RANKL expressed on the surface of osteoblasts (OB) or bone marrow stromal cells (BMSC) interacts with a cell surface receptor, RANK, present on pre-OC (induced by M-CSF) and mature OC to stimulate their fusion, development, bone resorption, and cell survival (5Lacey D. Timms E. Tan H.-L. Kelley M. Dunstan C. Burgess T. Elliott R. Colombero A. Elliott G. Scully S. Hsu H. Sullivan J. Hawkins N. Davy E. Capparelli C. Eli A. Qian Y.-X. Kaufman S. Sarosi I. Shalhoub V. Senaldi G. Guo J. Delaney J. Boyle W. Cell. 1998; 93: 165-176Abstract Full Text Full Text PDF PubMed Scopus (4656) Google Scholar, 6Yasuda H. Shima N. Nakagawa N. Yamaguchi K. Kinosaki M. Mochizuki S.-I. Tomoyasu A. Yano K. Goto M. Murakami A. Takahashi N. Suda T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3597-3602Crossref PubMed Scopus (3592) Google Scholar, 7Matsuzaki K. Udagawa N. Takahasi N. Yamaguchi K. Yasuda H. Shima N. Morinaga T. Toyama Y. Yabe Y. Higashio K. Suda T. Biochem. Biophys. Res. Commun. 1998; 246: 199-204Crossref PubMed Scopus (322) Google Scholar, 8Nakagawa N. Kinosaki M. Yamaguchi K. Shima N. Yasuda H. Yano K. Morinaga T. Higashio K. Biochem. Biophys. Res. Commun. 1998; 253: 395-400Crossref PubMed Scopus (628) Google Scholar, 9Hsu H. Lacey D. Dunstan C. Solovyev I. Colombero A. Timms E. Tan H.-L. Elliott G. Kelley M. Sarosi I. Wang L. Xia X.-Z. Elliott R. Chiu L. Black T. Scully S. Capparelli C. Morony S. Shimamoto G. Bass M. Boyle W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3540-3545Crossref PubMed Scopus (1428) Google Scholar). RANKL expression increases during early OB development and is up-regulated in OB and BMSC by various pro-resorptive stimuli such as parathyroid hormone (PTH), 1,25-dihydroxyvitamin D3(VD3), dexamethasone (Dex), prostaglandin E2, or interleukin-11 (IL-11) (6Yasuda H. Shima N. Nakagawa N. Yamaguchi K. Kinosaki M. Mochizuki S.-I. Tomoyasu A. Yano K. Goto M. Murakami A. Takahashi N. Suda T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3597-3602Crossref PubMed Scopus (3592) Google Scholar, 10Tsukii K. Shima N. Mochizuki S. Yamaguchi K. Kinosaki M. Yano K. Shibata O. Udagawa N. Yasuda H. Suda T. Higashio K. Biochem. Biophys. Res. Commun. 1998; 246: 337-341Crossref PubMed Scopus (221) Google Scholar, 11Nagai M. Sata N. Biochem. Biophys. Res. Commun. 1999; 257: 719-723Crossref PubMed Scopus (94) Google Scholar, 12Fuller K. Wong B. Fox S. Choi Y. Chambers T. J. Exp. Med. 1998; 188: 997-1001Crossref PubMed Scopus (508) Google Scholar). Recently, the pro-resorptive inflammatory cytokines TNF-α and IL-1β were also shown to elevate RANKL mRNA levels in human BMSC and MG63 osteosarcoma cells (13Hofbauer L. Lacey D. Dunstan C. Spelsberg T. Riggs B. Khosla S. Bone ( NY ). 1999; 25: 255-259Crossref PubMed Scopus (549) Google Scholar). Targeted ablation of RANKL in mice results in suppressed osteoclastogenesis and an osteopetrotic phenotype (14Kong Y. Yoshida H. Sarosi I. Tan H. Timms E. Capparelli C. Morony S. Oliveira-dos Santos A. Van G. Itie A. Khoo W. Wakeham A. Dunstan C. Lacey D. Mak T. Boyle W. Penninger J. Nature. 1999; 397: 315-323Crossref PubMed Scopus (2887) Google Scholar), whereas RANKL administration into normal adult mice elicits increased OC size and activation (but not numbers) and systemic hypercalcemia (5Lacey D. Timms E. Tan H.-L. Kelley M. Dunstan C. Burgess T. Elliott R. Colombero A. Elliott G. Scully S. Hsu H. Sullivan J. Hawkins N. Davy E. Capparelli C. Eli A. Qian Y.-X. Kaufman S. Sarosi I. Shalhoub V. Senaldi G. Guo J. Delaney J. Boyle W. Cell. 1998; 93: 165-176Abstract Full Text Full Text PDF PubMed Scopus (4656) Google Scholar). Osteoprotegerin (OPG, also known as osteoclastogenesis inhibitory factor or OCIF) is a naturally occurring soluble member of the TNF receptor superfamily that is widely expressed in multiple tissues and binds to RANKL, thereby neutralizing its function (1Hofbauer L. Khosla S. Dunstan C. Lacey D. Boyle W. Riggs B. J. Bone Miner. Res. 2000; 15: 2-12Crossref PubMed Scopus (1032) Google Scholar, 2Filvaroff E. Derynck R. Curr. Biol. 1998; 8: R679-R682Abstract Full Text Full Text PDF PubMed Google Scholar, 3Suda T. Takahashi N. Udagawa N. Jimi E. Gillespie M. Martin T. Endocr. Rev. 1999; 20: 345-357Crossref PubMed Google Scholar, 4Wong B. Josien R. Choi Y. J. Leukocyte Biol. 1999; 65: 715-724Crossref PubMed Scopus (199) Google Scholar). OPG therefore acts as a secreted decoy receptor to negatively regulate OC differentiation, activity, and survival both in vivo andin vitro (15Simonet W. Lacey D. Dunstan C. Kelley M. Chang M.-S. Luthy R. Nguyen H. Wooden S. Bennett L. Boone T. Shimamoto G. DeRose M. Elliott R. Colombero A. Tan H.-L. Trail G. Sullivan J. Davy E. Bucay N. Renshaw-Gegg L. Hughes T. Hill D. Pattison W. Campbell P. Sander S. Van G. Tarpley J. Derby P. Lee R. Amgen EST Program Boyle W. Cell. 1997; 89: 309-319Abstract Full Text Full Text PDF PubMed Scopus (4374) Google Scholar, 16Yasuda H. Shima N. Nakagawa N. Mochizuki S.-I. Yano K. Fujise N. Sato Y. Goto M. Yamaguchi K. Kuriyama M. Kanno T. Murakami A. Tsuda E. Morinaga T. Higashio K. Endocrinology. 1998; 139: 1329-1337Crossref PubMed Scopus (965) Google Scholar). OPG production by OB and BMSC is regulated by calcitropic hormones and cytokines, and the balance in the ratio of RANKL to OPG critically determines net effects on OC development and bone resorption (1Hofbauer L. Khosla S. Dunstan C. Lacey D. Boyle W. Riggs B. J. Bone Miner. Res. 2000; 15: 2-12Crossref PubMed Scopus (1032) Google Scholar, 2Filvaroff E. Derynck R. Curr. Biol. 1998; 8: R679-R682Abstract Full Text Full Text PDF PubMed Google Scholar, 3Suda T. Takahashi N. Udagawa N. Jimi E. Gillespie M. Martin T. Endocr. Rev. 1999; 20: 345-357Crossref PubMed Google Scholar, 4Wong B. Josien R. Choi Y. J. Leukocyte Biol. 1999; 65: 715-724Crossref PubMed Scopus (199) Google Scholar, 11Nagai M. Sata N. Biochem. Biophys. Res. Commun. 1999; 257: 719-723Crossref PubMed Scopus (94) Google Scholar). In vivoadministration of OPG to normal rats reduces osteoclastogenesis and increases bone density, OPG prevents estrogen deficiency-associated bone loss in ovariectomized animals, and transgenic mice overexpressing OPG exhibit increased bone density and severe osteopetrosis (15Simonet W. Lacey D. Dunstan C. Kelley M. Chang M.-S. Luthy R. Nguyen H. Wooden S. Bennett L. Boone T. Shimamoto G. DeRose M. Elliott R. Colombero A. Tan H.-L. Trail G. Sullivan J. Davy E. Bucay N. Renshaw-Gegg L. Hughes T. Hill D. Pattison W. Campbell P. Sander S. Van G. Tarpley J. Derby P. Lee R. Amgen EST Program Boyle W. Cell. 1997; 89: 309-319Abstract Full Text Full Text PDF PubMed Scopus (4374) Google Scholar). Conversely, mice deficient in OPG display increased OC development and activity, early onset osteoporosis, and arterial calcification (17Bucay N. Sarosi I. Dunstan C. Morony S. Tarpley J. Capparelli C. Scully S. Tan H. Xu W. Lacey D. Boyle W. Genes Dev. 1998; 12: 1260-1268Crossref PubMed Scopus (2144) Google Scholar,18Mizuno A. Amizuka N. Irie K. Murakami A. Fujise N. Kanno T. Sato Y. Nakagawa N. Yasuda H. Mochizuki S.-I. Gomibuchi T. Yano K. Shima N. Washida N. Tsuda E. Morinaga T. Higashio K. Ozawa H. Biochem. Biophys. Res. Commun. 1998; 247: 610-615Crossref PubMed Scopus (709) Google Scholar). Thus, OPG has been proposed to regulate bone resorption both locally and systemically, as well as to serve as a physiological suppressive signal of local calcification in blood vessels (2Filvaroff E. Derynck R. Curr. Biol. 1998; 8: R679-R682Abstract Full Text Full Text PDF PubMed Google Scholar, 17Bucay N. Sarosi I. Dunstan C. Morony S. Tarpley J. Capparelli C. Scully S. Tan H. Xu W. Lacey D. Boyle W. Genes Dev. 1998; 12: 1260-1268Crossref PubMed Scopus (2144) Google Scholar). Vascular endothelial cells (VEC) are intimately associated with pre-OC and OC during both their formation and resorption of bone (19Collin-Osdoby P. J. Cell. Biochem. 1994; 55: 304-309Crossref PubMed Scopus (147) Google Scholar, 20Streeten E. Brandi M. Bone Miner. 1990; 10: 85-94Abstract Full Text PDF PubMed Scopus (81) Google Scholar, 21Burkhardt B. Kettner G. Bohm W. Schmidmeier M. Schlag R. Frisch B. Mallmann B. Eisenmenger W. Gilg T. Bone ( NY ). 1987; 8: 157-164Crossref PubMed Scopus (375) Google Scholar, 22Gerber H.-P. Vu T. Ryan A. Kowalski J. Werb Z. Ferrara N. Nat. Med. 1999; 5: 623-628Crossref PubMed Scopus (1723) Google Scholar). This close physical interaction permits VEC to influence directly and convey local and systemic regulatory signals for OC development and bone remodeling under normal or pathological conditions (19Collin-Osdoby P. J. Cell. Biochem. 1994; 55: 304-309Crossref PubMed Scopus (147) Google Scholar, 20Streeten E. Brandi M. Bone Miner. 1990; 10: 85-94Abstract Full Text PDF PubMed Scopus (81) Google Scholar, 21Burkhardt B. Kettner G. Bohm W. Schmidmeier M. Schlag R. Frisch B. Mallmann B. Eisenmenger W. Gilg T. Bone ( NY ). 1987; 8: 157-164Crossref PubMed Scopus (375) Google Scholar, 22Gerber H.-P. Vu T. Ryan A. Kowalski J. Werb Z. Ferrara N. Nat. Med. 1999; 5: 623-628Crossref PubMed Scopus (1723) Google Scholar). Recent studies indicate that many pre-OCs reside in the peripheral circulation as well as in the bone marrow (23Parfitt A. Bone ( NY ). 1998; 23: 491-494Crossref PubMed Scopus (66) Google Scholar, 24Faust J. Lacey D. Hunt P. Burgess T. Scully S. Van G. Eli A. Qian Y. Shalhoub V. J. Cell. Biochem. 1999; 72: 67-80Crossref PubMed Scopus (88) Google Scholar, 25Wani M. Fuller K. Kim N. Choi Y. Chambers T. Endocrinology. 1999; 140: 1927-1935Crossref PubMed Google Scholar, 26Shalhoub V. Faust J. Boyle W. Dunstan C. Kelley M. Kaufman S. Scully S. Van G. Lacey D. J. Cell. Biochem. 1999; 72: 251-261Crossref PubMed Scopus (114) Google Scholar). Circulating pre-OC may therefore also be exposed to and potentially activated by signaling molecules displayed on the VEC surface during and following their transmigration across the VEC layer of blood vessels in response to local stimulatory signals to reach the bone microenvironment. Although all OCs develop and function in close association with the VEC and sinusoids of bone, specific signals mediating interactions between these cells are not well known. Because VEC function as primary immune response cells that are potently activated by TNF-α and IL-1 (27Cines D. Pollak E. Buck C. Loscalzo J. Zimmerman G. McEver R. Pober J. Wick T. Konkle B. Schwartz B. Barnathan E. McCrae K. Hug B. Schmidt A. Stern D. Blood. 1998; 91: 3527-3561PubMed Google Scholar,28Mantovani A. Bussolino F. Dejana E. FASEB J. 1992; 6: 2591-2599Crossref PubMed Scopus (629) Google Scholar), we investigated whether primary human VEC expressed RANKL and/or OPG, and if such expression was regulated by these or other pro-resorptive stimuli. The functional consequences of RANKL expression and regulation in HMVEC were assessed relative to the in vitro development and activity of multinucleated bone-resorptive OCs from precursors present in human peripheral blood mononuclear cell preparations. Primary HMVEC of normal adult female dermal tissue origin, media (essential growth medium-microvascular, EGM-MV, and essential basal medium, EBM), and media supplements (packaged as SingleQuots containing human recombinant epidermal growth factor, hydrocortisone, gentamicin, bovine brain extract, and fetal bovine serum, FBS) were obtained from Clonetics Corp. (San Diego, CA). HMVEC were grown, subcultured by trypsin/EDTA, and used within 4 passages as recommended. HMVEC expressed all the hallmark characteristics of endothelial cells (morphology, tubule formation, acetylated low density lipoprotein uptake, factor VIII expression, PECAM-1, ICAM-1, VCAM-1, ELAM-1, P-selectin, ACE, vimentin, and no smooth muscle α-actin). For molecular analyses, HMVEC were cultured to near confluence in 24-well dishes in EGM-MV complete medium (with supplements and 5% FBS); modulators were administered the next day in fresh medium, and the cells were incubated for the times indicated before RNA was harvested. For withdrawal experiments, the modulator medium was removed after the induction period, the cells were rinsed twice briefly with fresh medium, and the cells were cultured in medium without modulator for various times before RNA was harvested. Cytokine release by HMVEC was evaluated in cells grown to near confluence in EGM-MV complete medium and switched to phenol red-free EBM (lacking supplements) plus 5% FBS for 16–24 h before modulators were administered in fresh medium. The conditioned medium (briefly centrifuged) and cells were harvested after 24 h and stored at −80 °C until analyzed for cytokine and protein levels, respectively. Modulators used were 1,25-dihydroxyvitamin D3 (VD3, a gift of Hoffmann-La Roche), dexamethasone (Dex), and human parathyroid hormone-(1–34) (PTH-(1–34); both from Sigma), and human recombinant cytokines TNF-α, IL-1α, and M-CSF (all from R & D Systems, Minneapolis, MN). Primary human osteoblasts (HOB) were obtained as cultured outgrowth cells from trabecular bone explants according to the Robey/Termine method as described previously (29Chaudhary L. Cheng S.-L. Avioli L. Mol. Cell. Biochem. 1996; 156: 69-77Crossref PubMed Google Scholar). Human bone marrow stromal cells (HBMSC) were isolated from discarded thoracic rib surgical specimens or bone obtained from accident victims and cultured in α-MEM with 10% FBS and 1% antibiotic/antimycotic (Life Technologies, Inc.), with or without Dex (100 nm) and VD3 (10 nm) to promote a more OB-like phenotype as described previously (29Chaudhary L. Cheng S.-L. Avioli L. Mol. Cell. Biochem. 1996; 156: 69-77Crossref PubMed Google Scholar, 30Cheng S.-L. Yang J. Rifas L. Zhang S.-F. Avioli L. Endocrinology. 1994; 134: 277-286Crossref PubMed Scopus (529) Google Scholar). RNA was isolated from cells using RNA STAT-60 (Tel-Test, Inc., Friendswood, TX). Semi-quantitative RT-PCR amplification for RANKL was performed using forward and reverse primers (designed by Drs. N. Weitzmann and L. Rifas, Washington University, St. Louis, MO) to the extracellular region of OPGL/TRANCE/RANKL (nucleotides 4–735) of cloned human TRANCE (nucleotides 1–738, GenBankTM accession number AF013171) and Amersham Pharmacia Biotech Ready-to-Go RT-PCR beads. Oligonucleotide primers were as follows: forward, 5′-GCTCTAGAGCCATGGATCCTAATAGAAT-3′, and reverse 5′-ATCTCGAGTCACTATTAATGATGATGATGATGATGATCTATATCTCGAACTTTAAAAGCC-3′. RT-PCR amplification for OPG was performed using forward and reverse primers to cloned human OPG (GenBankTM accession numberU94332) as follows: forward, 5′-GGGGACCACAATGAACAAGTTG-3′ (nucleotides 85–106), and reverse, 5′-AGCTTGCACCACTCCAAATCC-3′ (nucleotides 473–493) (31Thomas R. Guise T. Yin J. Elliott J. Horwood N. Martin T. Gillespie M. Endocrinology. 1999; 140: 4451-4458Crossref PubMed Google Scholar). Parallel reactions were performed for every assay using primers designed to amplify human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as described previously (32Sunyer T. Lewis J. Collin-Osdoby P. Osdoby P. J. Clin. Invest. 1999; 103: 1409-1418Crossref PubMed Scopus (124) Google Scholar). Initial trials were performed using RNA obtained from unstimulated cultures as well as each modulator treatment to establish optimal cycle numbers and RNA amounts for routine use so that RT-PCRs would yield RANKL, OPG, and GAPDH amplifications within an exponentially linear range over the amount of input RNA used. Thus, cycle numbers were varied from 20 to 35 (OPG), 30 to 35 (RANKL), and 15 to 35 (GAPDH), and RNA amounts from 0.15 ng to 1.5 μg (OPG), 25 ng to 12 μg (RANKL), and 1 pg to 4 μg (GAPDH), in up to 6 replicate trials each. No products were obtained in controls lacking either RNA or first strand primers, and amplified products were not eliminated or reduced by DNase treatment of the RNA samples. Conditions were chosen that consistently provided mid-linear range amplification of each PCR product for all further studies. Therefore, PCRs were run at 94 °C for 1 min, 60 °C for 1 min, and 72 °C for 2 min for 35 (RANKL), 26 (OPG), or 20 (GAPDH) cycles. Products were separated by agarose gel electrophoresis, visualized by ethidium bromide staining, photographed using a Polaroid camera, and quantified in a scanner (Scanjet II, Hewlett-Packard) computer linked to a Quantimet image analysis system (Leica, United Kingdom). RANKL or OPG signals were normalized to GAPDH signals determined in parallel for each sample, and data were expressed as a percentage of the RANKL/GAPDH or OPG/GAPDH ratio for untreated HMVEC measured in the same trial. The 731-bp RANKL and 408-bp OPG amplicons generated by RT-PCR from HMVEC (as well as HOB and HBMSC) were directly sequenced using an ABI Prism Cycle Sequencing kit (PerkinElmer Life Sciences), and DNA sequences were compared with published sequences to confirm their identity using computation performed at the NCBI and the BLAST network service. Cytokine levels in conditioned medium were measured using specific enzyme-linked immunoassay kits (Quantikine kits, R & D Systems, Minneapolis, MN) for human IL-1β, TNF-α, and M-CSF as recommended. Standard curves were run with each assay, and control medium was analyzed for background levels of each cytokine (which were insignificant), and modulators were tested in at least 3 separate culture wells per trial for 2–8 independent HMVEC cultures. Results were normalized for cell protein using the bicinchoninic acid protein assay (Pierce) and bovine serum albumin (BSA) as a standard (33Rothe L. Collin-Osdoby P. Chen Y. Sunyer T. Chaudhary L. Tsay A. Goldring S. Avioli L. Osdoby P. Endocrinology. 1998; 139: 4353-4363Crossref PubMed Google Scholar), and data were expressed as the mean ± S.E. ng/ml of cytokine released per mg of cell protein during 24 h of culture. HMVEC were cultured in EGM-MV complete medium on glass coverslips in 24-well tissue culture dishes to near confluency, TNF-α (1 nm) was administered in fresh medium for 24 h, and the cells were fixed and immunostained (34Collin-Osdoby P. Oursler M. Rothe L. Webber D. Anderson F. Osdoby P. J. Bone Miner. Res. 1995; 10: 45-58Crossref PubMed Scopus (20) Google Scholar). Briefly, HMVEC were rinsed, fixed in 3% paraformaldehyde/HBSS (15 min), rinsed, blocked for 1 h with 1% BSA and 10% horse serum in phosphate-buffered saline (PBS), and reacted for 1 h with (or without) primary antibodies diluted in block. Mouse monoclonal antibodies (mAb) specific for human ICAM-1 or VCAM-1 (Serotec, Raleigh, NC, each at 1:200 dilution), mAb to the angiogenesis-related integrin αvβ3 (LM 609, Chemicon International, Temecula, CA, 1:100 dilution), and goat polyclonal antibody (pAb) raised to a C-terminal extracellular peptide region of human RANKL (Santa Cruz Biotechnology, Santa Cruz, CA, 1:100 dilution) were used. Primary antibody binding was detected using a secondary goat anti-mouse fluorescein isothiocyanate conjugate (Life Technologies, Inc.; 1:200) for mAbs or a biotinylated donkey anti-goat antibody (Santa Cruz Biotechnology; 1:200) followed by a streptavidin-Texas Red conjugate (Life Technologies, Inc.; 1:1000) for RANKL pAb. In some cases, HMVEC were simultaneously immunostained for both ICAM-1 and RANKL to visualize their co-localization on the HMVEC plasma membrane. Coverslips were mounted on glass microscope slides in glycerol-buffered mounting medium (Becton Dickinson, Cockeysville, MD), and images were viewed and digitally captured using a Leica scanning laser (argon/krypton/He) confocal microscope (TCS-SP-2) equipped with a 20× phase objective. Wavelengths for excitation and emission for fluorescein isothiocyanate were 494 and 518 nm, respectively, and those for Texas Red were 595 and 615 nm, respectively. Human osteoporotic bone was obtained from fractured femoral heads discarded during hip replacement surgery and briefly held at 4 °C in α-MEM before fixation in 10% buffered formalin. Samples were decalcified, paraffin-embedded, and sectioned by standard procedures. Sections were prepared for immunostaining by deparaffinization in xylene, hydration through 100% EtOH, 95% EtOH, and water, and heating (97 °C for 20 min) in an antigen unmasking Target Retrieval Solution (Dako, Carpinteria, CA). Cooled sections were PBS-rinsed, endogenous peroxidase activity was quenched in Dako Peroxidase Blocking reagent (15 min), rinsed sections were blocked with Dako serum-free Protein Block (10 min), and sections were reacted overnight at 4 °C with the pAb to human RANKL described above (diluted 1:500 to 1:1000 in PBS + 1.5% Dako Protein Block). Sections were rinsed, incubated with biotinylated donkey anti-goat pAb (Santa Cruz Biotechnology, 1:100 dilution in PBS/block, 45 min) followed by Dako streptavidin-peroxidase (1:300 in PBS, 15 min), reacted with Dako diaminobenzidine solution (5 min), briefly counterstained using Dako hematoxylin solution, and mounted on glass slides with Permount. Immunostained sections were viewed by light microscopy, and images were digitally captured using a computer-linked Olympus microscope. HMVEC were cultured in 24-well dishes in EGM complete medium to near confluency, two-thirds of the wells were treated for 48 h with either 1 nm TNF-α or IL-1α to maximally induce RANKL (while allowing stimulated OPG levels to decline), and the cells were washed (3 times) to remove cytokines just prior to the addition of human PBMC for co-culture. Human PBMC were prepared from heparinized blood obtained from the American Red Cross (St. Louis, MO). Mononuclear cells were isolated by Ficoll-Paque (26Shalhoub V. Faust J. Boyle W. Dunstan C. Kelley M. Kaufman S. Scully S. Van G. Lacey D. J. Cell. Biochem. 1999; 72: 251-261Crossref PubMed Scopus (114) Google Scholar,32Sunyer T. Lewis J. Collin-Osdoby P. Osdoby P. J. Clin. Invest. 1999; 103: 1409-1418Crossref PubMed Scopus (124) Google Scholar, 33Rothe L. Collin-Osdoby P. Chen Y. Sunyer T. Chaudhary L. Tsay A. Goldring S. Avioli L. Osdoby P. Endocrinology. 1998; 139: 4353-4363Crossref PubMed Google Scholar), resuspended in α-MEM plus 10% FBS and 1% antibiotic/antimycotic, and added (1.6 × 106PBMC/well) to the 24-well dish containing unactivated or cytokine-pre-activated HMVEC. Some wells also received 100 ng/ml recombinant human OPG:Fc fusion peptide (Alexis Corporation, San Diego, CA). The next day (day 1) all wells were treated with 10 nmVD3 and 25 ng/ml M-CSF, and OPG:Fc was readministered to the wells originally receiving this treatment. On day 5 the cells were refed with M-CSF, with or without OPG:Fc, and the cells were harvested on day 7, rinsed, fixed in 3% PF/HBSS, rinsed, and stained for TRAP activity (34Collin-Osdoby P. Oursler M. Rothe L. Webber D. Anderson F. Osdoby P. J. Bone Miner. Res. 1995; 10: 45-58Crossref PubMed Scopus (20) Google Scholar, 35Kasten T. Collin-Osdoby P. Patel N. Osdoby P. Krukowski M. Misko T. Settle S. Currie M. Nickols G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3569-3573Crossref PubMed Scopus (232) Google Scholar, 36Collin-Osdoby P. Rothe L. Bekker S. Anderson F. Osdoby P. J. Bone Miner. Res. 2000; 15: 474-488Crossref PubMed Scopus (61) Google Scholar). Cells were co-stained with" @default.
• W2060482009 created "2016-06-24" @default.
• W2060482009 creator A5024516865 @default.
• W2060482009 creator A5038638225 @default.
• W2060482009 creator A5043611817 @default.
• W2060482009 creator A5050470911 @default.
• W2060482009 creator A5065335405 @default.
• W2060482009 creator A5070026560 @default.
• W2060482009 date "2001-01-01" @default.
• W2060482009 modified "2023-10-17" @default.
• W2060482009 title "Receptor Activator of NF-κB and Osteoprotegerin Expression by Human Microvascular Endothelial Cells, Regulation by Inflammatory Cytokines, and Role in Human Osteoclastogenesis" @default.
• W2060482009 cites W1510237891 @default.
• W2060482009 cites W1534645474 @default.
• W2060482009 cites W1583699618 @default.
• W2060482009 cites W1587762493 @default.
• W2060482009 cites W1937290251 @default.
• W2060482009 cites W1971017689 @default.
• W2060482009 cites W1971720206 @default.
• W2060482009 cites W1974659965 @default.
• W2060482009 cites W1977013832 @default.
• W2060482009 cites W1982011044 @default.
• W2060482009 cites W1991730557 @default.
• W2060482009 cites W2000647744 @default.
• W2060482009 cites W2003733256 @default.
• W2060482009 cites W2003865675 @default.
• W2060482009 cites W2010359869 @default.
• W2060482009 cites W2013762025 @default.
• W2060482009 cites W2016182569 @default.
• W2060482009 cites W2018602999 @default.
• W2060482009 cites W2025679272 @default.
• W2060482009 cites W2034711065 @default.
• W2060482009 cites W2035086883 @default.
• W2060482009 cites W2035087930 @default.
• W2060482009 cites W2036165258 @default.
• W2060482009 cites W2037401343 @default.
• W2060482009 cites W2040873162 @default.
• W2060482009 cites W2044192395 @default.
• W2060482009 cites W2046390154 @default.
• W2060482009 cites W2047380663 @default.
• W2060482009 cites W2048299560 @default.
• W2060482009 cites W2050567975 @default.
• W2060482009 cites W2058262190 @default.
• W2060482009 cites W2063048904 @default.
• W2060482009 cites W2068937569 @default.
• W2060482009 cites W2074007657 @default.
• W2060482009 cites W2085755629 @default.
• W2060482009 cites W2092320322 @default.
• W2060482009 cites W2093147348 @default.
• W2060482009 cites W2105332564 @default.
• W2060482009 cites W2106766464 @default.
• W2060482009 cites W2107925338 @default.
• W2060482009 cites W2109608328 @default.
• W2060482009 cites W2114216625 @default.
• W2060482009 cites W2124036292 @default.
• W2060482009 cites W2129651548 @default.
• W2060482009 cites W2132646563 @default.
• W2060482009 cites W2133694413 @default.
• W2060482009 cites W2136257629 @default.
• W2060482009 cites W2139996507 @default.
• W2060482009 cites W2153644776 @default.
• W2060482009 cites W2156467557 @default.
• W2060482009 cites W2160578379 @default.
• W2060482009 cites W2161919224 @default.
• W2060482009 cites W2167886526 @default.
• W2060482009 cites W2341093174 @default.
• W2060482009 doi "https://doi.org/10.1074/jbc.m010153200" @default.
• W2060482009 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/11274143" @default.
• W2060482009 hasPublicationYear "2001" @default.
• W2060482009 type Work @default.
• W2060482009 sameAs 2060482009 @default.
• W2060482009 citedByCount "383" @default.
• W2060482009 countsByYear W20604820092012 @default.
• W2060482009 countsByYear W20604820092013 @default.
• W2060482009 countsByYear W20604820092014 @default.
• W2060482009 countsByYear W20604820092015 @default.
• W2060482009 countsByYear W20604820092016 @default.
• W2060482009 countsByYear W20604820092017 @default.
• W2060482009 countsByYear W20604820092018 @default.
• W2060482009 countsByYear W20604820092019 @default.
• W2060482009 countsByYear W20604820092020 @default.
• W2060482009 countsByYear W20604820092021 @default.
• W2060482009 countsByYear W20604820092022 @default.
• W2060482009 countsByYear W20604820092023 @default.
• W2060482009 crossrefType "journal-article" @default.
• W2060482009 hasAuthorship W2060482009A5024516865 @default.
• W2060482009 hasAuthorship W2060482009A5038638225 @default.
• W2060482009 hasAuthorship W2060482009A5043611817 @default.
• W2060482009 hasAuthorship W2060482009A5050470911 @default.
• W2060482009 hasAuthorship W2060482009A5065335405 @default.
• W2060482009 hasAuthorship W2060482009A5070026560 @default.
• W2060482009 hasBestOaLocation W20604820091 @default.
• W2060482009 hasConcept C164027704 @default.
• W2060482009 hasConcept C170493617 @default.
• W2060482009 hasConcept C185592680 @default.
• W2060482009 hasConcept C203014093 @default.
• W2060482009 hasConcept C2776914184 @default.
• W2060482009 hasConcept C2777730290 @default.
• W2060482009 hasConcept C2779428903 @default.

• next