Proteoglycan 4, a Novel Immunomodulatory Factor, Regulates Parathyroid Hormone Actions on Hematopoietic Cells

• DOI: 10.1016/j.ajpath.2011.07.032
• Publication Date: 2011-09-22T12:56:17Z
• Authors: Chad M. Novince; Amy J. Koh; Megan N. Michalski; Julie T. Marchesan; Jason Wang; Younghun Jung; Janice E. Berry; Matthew R. Eber; Thomas J. Rosol; Russell S. Taichman; Laurie K. McCauley
• Keywords: 0301 basic medicine; Interleukin-6; Hematopoietic Stem Cells; Chemokine CXCL12; Hematopoiesis; 3. Good health; Mice, Inbred C57BL; Mice; 03 medical and health sciences; Thrombopoietin; Bone Marrow; Parathyroid Hormone; Animals; Proteoglycans; Lymphocytes; RNA, Messenger; Megakaryocyte Progenitor Cells

Proteoglycan 4 (PRG4), a critical protective factor in articular joints, is implicated hematopoietic progenitor cell expansion and megakaryopoiesis. PRG4 loss-of-function mutations result camptodactyly-arthropathy-coxa vara-pericarditis (CACP) syndrome, which characterized primarily by precocious joint failure. was identified as novel parathyroid hormone (PTH) responsiveness gene osteoblastic cells bone, investigated potential mediator of PTH actions on hematopoiesis. Sixteen-week-old Prg4−/− mutant Prg4+/+ wild-type mice were treated daily with intermittent (residues 1–34) or vehicle for 6 weeks. At 22 weeks age, Prg4 had increased peripheral blood neutrophils decreased marrow B220+ (B-lymphocytic) cells, normalized PTH. The PTH-induced increase Lin−Sca-1+c-Kit+ (hematopoietic progenitor) blunted mice. Basal PTH-stimulated stromal cell-derived factor-1 (SDF-1) mice, suggesting SDF-1 candidate regulator proteoglycan hematopoiesis vivo. stimulation IL-6 mRNA greater than calvaria bone marrow, compensatory mechanism the cells. In summary, PTH-responsive regulating immune Parathyroid (PTH), best known its roles calcium homeostasis remodeling,1Kousteni S. Bilezikian J.P. Cellular hormone.in: Raisz L.G. Martin T.J. Principles Bone Biology. ed 3. 2008: 639-656Crossref Scopus (19) Google Scholar supports (HPC) mobilization.2Calvi L.M. Adams G.B. Welbrecht K.W. Weber J.M. Olson D.P. Knight M.C. R.P. Schipani E. Divieti P. Bringhurst F.R. Milner L.A. Kronenberg H.M. Scadden D.T. Osteoblastic regulate haematopoietic stem niche.Nature. 2003; 425: 841-846Crossref PubMed (2798) Scholar, 3Brunner Zaruba M. Huber B. David R. Vallaster Assmann G. Mueller-Hoecker J. 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Berry Ghaname Kamarajan Bonnelye Ross C.W. Kapila Jurdic McCauley mediates interleukin-6.PLoS One. 2010; 5: e13657Crossref (30) using global knockout mouse model (human PRG4; Prg4), has been expansion14Liu Y.J. Lu S.H. Xu R.C. Ren Yan F.Y. Han Z.B. Z.C. Hemangiopoietin, growth primitive both endothelial lineages.Blood. 2004; 103: 4449-4456Crossref (58) 15Xu Z.L. Zhou Cong X.L. Y.H. Gu Hemangiopoietin animal survival accelerates recovery chemotherapy-suppressed mice.Eur J Haematol. 79: 477-485Crossref (4) 16Xu Z.S. Lv L.L. He Wang T. Chen Z.Z. transduced hemangiopoietin support vitro.Haematologica. 2005; 90: 157-165PubMed megakaryopoiesis,17Merberg D.M. Fitz L.J. Temple Giannotti Murtha Fitzgerald Scaltreto Kelleher Preissner Kriz Jacobs A comparison vitronectin megakaryocyte stimulating factor.in: Rosenblatt Kost C. Wegerhoff Mosher D.F. Biology Vitronectins Their Receptors: Proceedings First International Vitronectin Workshop, Rauischholzhausen Castle, Marburg, Germany, 25–28 August, 1993. Elsevier, Philadelphia1992: 45-53Google 18Greenberg S.M. Kuter D.J. Rosenberg R.D. vitro maturation stimulatory factor.J 1987; 262: 3269-3277Abstract 19Tayrien Purification properties present serum-free conditioned medium embryonic kidney thrombocytopenic plasma.J 3262-3268Abstract bone.20Novince C.M. Marchesan J.T. Proteoglycan-4: anabolism (abstract 1186) ASBMR 31st Annual Meeting 1001–1300.J 24: S56-S57Google 21Novince Sinder B.P. Entezami Eber M.J. Pettway G.J. Wronski Kozloff K.M. 4: dynamic skeletogenesis anabolism.J 2011https://doi.org/10.1002/jbmr.508Google encodes an approximately 345-kDa proteoglycan, consisting 1404 amino acids spanning 12 exons. expressed across nonskeletal tissues, highest relative levels liver, bone.22Ikegawa Sano Koshizuka Y. Nakamura Isolation, characterization mapping (proteoglycan 4) genes.Cytogenet Genome 2000; 291-297Crossref (139) 23Rhee D.K. Marcelino Baker Gong Smits Lefebvre V. Jay G.D. Stewart Warman M.L. Carpten J.D. secreted glycoprotein lubricin protects surfaces synovial overgrowth.J Clin Invest. 115: 622-631Crossref (421) four isolated products [ie, lubricin, superficial zone (SZP), (HAPO), (MSF)] glycoproteins protection,23Rhee 24Schumacher Block Schmid T.M. Aydelotte M.B. Kuettner K.E. synthesized chondrocytes cartilage.Arch Biochem Biophys. 1994; 311: 144-152Crossref (302) expansion,14Liu megakaryopoiesis17Merberg (Table 1). not yet identified.14Liu Loss-of-function autosomal recessive disorder syndrome (CACP), early-onset failure osteopenia. CACP patients reported spontaneously develop hematological disorders.25Marcelino Suwairi Gutierrez O.M. Schwartz Robbins Sood Makalowska I. Baxevanis A. Johnstone Laxer Zemel Kim C.A. Herd J.K. Ihle Williams Johnson Raman Alonso Brunoni Gerstein Papadopoulos Bahabri S.A. Trent CACP, encoding mutated syndrome.Nat Genet. 23: 319-322Crossref (237) ScholarTable 1PRG4 Protein ProductsProtein productIsolationExpressionKnown functionsLubricin23Rhee ScholarBovine fluidsSynoviocytesBoundary lubrication jointsSuperficial (SZP)24Schumacher explant culture mediaSuperficial chondrocytesProtects cartilageHemangiopoietin (HAPO)14Liu ScholarUrine afflicted aplastic anemiaUnknownSupports cellsMegakaryocyte (MSF)17Merberg transfer acute thrombocytopeniaUnknownRegulates megakaryopoiesis Open table new tab selected HAPO MSF regulates unclear. purpose study investigate unexplored physiological skeleton. Using Prg4-null model,23Rhee we report first C57BL/6 single subcutaneous injection recombinant residues 1 34 [PTH(1–34); μg/g] (Bachem, Torrance, CA) (0.9% NaCl) then sacrificed 4, 8, hours later; harvested analysis. All approved University Committee Use Care Animals (UCUCA) Michigan, animals maintained accordance UCUCA research protocols. Primary osteoblasts calvariae 7- 14-day-old Briefly, dissected, periosteum, subjected sequential digestion 10, 20, 60 minutes α-modified minimum essential (Invitrogen, Carlsbad, containing collagenase (2 mg/mL) (Roche Molecular Biochemicals, Indianapolis, IN) 0.25% trypsin (Invitrogen). Cells last digest washed, counted, plated at 5 × 104/cm2 10% fetal bovine serum (Invitrogen), 100 U/mL penicillin, mg/mL streptomycin, 2 mmol/L glutamine. induced differentiate supplementing 50 μg/mL ascorbic acid. Cultures day (d5) PTH(1–34) (10 nmol/L) (Bachem) (4 HCl/0.1%BSA) treatment 12, 24 later another experimental protocol, d1, d5, d11, d14 cultures allele generated homologous recombination 129Sv/Ev-derived generously provided Matthew (Harvard University).23Rhee Mutant backcrossed 129Sv/Ev genetic background background. PCR-based assay used genotype described previously.23Rhee 16 littermate (1 μg/g) 1, 16-week-old (50 μg/kg) control weeks, age. Mice final injection, tissues analyses. directly flushed femur tibia TRIzol reagent (Invitrogen) 3-mL syringe 22G ½-inch (∼12.7-mm length) needle. Calvariae flash-frozen, pulverized, homogenized reagent. Calvarial osteoblast washed three times 1× PBS, applied cultures. each case, RNA according manufacturer's directions, total quantified. Double-stranded cDNA 1.0 μg RNA, random hexamers MultiScribe reverse transcriptase (Applied Biosystems, Foster City, CA). amplified TaqMan universal PCR master mix expression-specific primers probes Biosystems) interleukin-6 (IL-6), thrombopoietin (TPO), (SDF-1), osteocalcin (OCN). Rodent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) endogenous control. Amplification performed ABI Prism 7700 sequence detection system Biosystems). Relative quantification data determined standard curve method comparative Ct method.26Schmittgen T.D. Livak K.J. Analyzing real-time CT method.Nat Protocols. 3: 1101-1108Crossref (17273) Whole collected cardiac puncture, transferred Microtainer tubes potassium-EDTA (BD Biosciences, San Jose, gently agitated. Complete count analysis (automated differential) Hemavet 1500FS (Drew Scientific, Waterbury, CT). Flushed femoral dissociated spleen isolated, all staining steps flow cytometry buffer (FCSB) (1× 2% serum, EDTA). Femurs sectioned mid-diaphysis, needle rotated epiphysis into cavity, out mid-diaphysis FCSB. Spleens incised lengthwise, placed strainer, disassociated 1-mL plunger while FCSB being delivered same time. suspensions homogenized, strained, centrifuged, resuspended; counts performed. Three million suspended 0.5 mL FCSB, incubated fluorochrome-conjugated antibodies Biosciences) 30 4°C, resuspended 7-AAD BD Biosciences. frequency CD11b+GR1+ (immature myeloid) CD3+ (T-lymphocytic) CD41+ (megakaryopoietic) assessed spleens. Samples analyzed FACSCalibur cytometer CellQuest Pro software version 5.1 Biosciences). double-staining technique assess ploidy. FITC-conjugated CD41 antibody rinsed fixed 1% paraformaldehyde overnight 4°C. propidium iodide solution sodium citrate, 0.1% Triton X-100, iodide, RNase A), hour 37°C. 10,000 gated sample. Tibiae phosphate-buffered formalin 48 dehydrated graded ethanols xylene, embedded modified methylmethacrylate. Frontal serial sections μm) cut vertical bed microtomes (Jung 2065 2165; Leica Microsystems, Bannockburn, IL) affixed slides precoated gelatin solution. Sections stained von Kossa tetrachrome counterstain (Polysciences, Warrington, PA), enumerate megakaryocytes per area. Histomorphometric semiautomatically Nikon light microscope OsteoMeasure trabecular (OsteoMetrics, Atlanta, GA). Tibiae, liver (left lobe), decalcified 14% EDTA 14 days room temperature. paraffin, 5-μm-thick stained. H&E tissue sections. immunofluorescence proximal deparaffinized mixed xylenes, rehydrated ethanols, briefly PBT (PBS-0.2% X-100) blocked Image-iT FX signal enhancer Zenon AlexaFluor 488 rabbit labeling kit α polyclonal (ab25117; Abcam, Cambridge, MA). After incubation 4°C μg/100 μL dilution fluorescence-labeled (Abcam), PBT, mounted ProLong Gold antifade DAPI coverslipped. Images acquired Olympus FV-500 confocal microscope. Image (secondary spongiosa) Plus (Media Cybernetics, Silver Spring, MD). 0.9% NaCl suspension centrifuged 81.6 g minutes, supernatants collected, stored −80°C, freeze-thawed once. supernatant sandwich