Bone Morphogenetic Proteins Are Overexpressed in the Bone Marrow of Primary Myelofibrosis and Are Apparently Induced by Fibrogenic Cytokines
2008; Elsevier BV; Volume: 172; Issue: 4 Linguagem: Inglês
10.2353/ajpath.2008.071030
ISSN1525-2191
AutoresOliver Bock, Julia Höftmann, Katharina Theophile, Kais Hussein, Birgitt Wiese, Jérôme Schlué, Hans Kreipe,
Tópico(s)Acute Myeloid Leukemia Research
ResumoPrimary myelofibrosis (PMF) is a myeloproliferative neoplasia characterized by progressive deposition of extracellular matrix components in the bone marrow. The involvement of members of the bone morphogenetic protein (BMP) family in aberrant bone marrow matrix homeostasis in PMF has not yet been investigated. Therefore, we analyzed expression of BMP1, an activator of latent transforming growth factor β-1 (TGFβ-1) and processor of collagen precursors, and other BMPs in bone marrow from PMF patients and controls (n = 95). Expression of BMP1, BMP6, BMP7, and BMP-receptor 2 was significantly increased in advanced stages of myelofibrosis compared with controls (P ≤ 0.01), and enhanced levels of BMP6 expression were already evident in prefibrotic stages of PMF. Immunohistochemistry showed that bone marrow stromal cells and megakaryocytes were the major cellular sources of BMP1 protein. Because TGFβ-1 and basic fibroblast growth factor have been shown to be important in the development of myelofibrosis, we studied the induction of BMPs by these cytokines in cultured fibroblasts. Fibroblasts treated with TGFβ-1 showed a pronounced up-regulation of BMP6, suggesting that stromal cells may be susceptible to BMP activation by cytokines with a proven role in the pathogenesis of PMF. We conclude that BMP family members may play an important role in the pathogenesis of myelofibrosis in PMF and are apparently induced by cytokines such as TGFβ-1. Primary myelofibrosis (PMF) is a myeloproliferative neoplasia characterized by progressive deposition of extracellular matrix components in the bone marrow. The involvement of members of the bone morphogenetic protein (BMP) family in aberrant bone marrow matrix homeostasis in PMF has not yet been investigated. Therefore, we analyzed expression of BMP1, an activator of latent transforming growth factor β-1 (TGFβ-1) and processor of collagen precursors, and other BMPs in bone marrow from PMF patients and controls (n = 95). Expression of BMP1, BMP6, BMP7, and BMP-receptor 2 was significantly increased in advanced stages of myelofibrosis compared with controls (P ≤ 0.01), and enhanced levels of BMP6 expression were already evident in prefibrotic stages of PMF. Immunohistochemistry showed that bone marrow stromal cells and megakaryocytes were the major cellular sources of BMP1 protein. Because TGFβ-1 and basic fibroblast growth factor have been shown to be important in the development of myelofibrosis, we studied the induction of BMPs by these cytokines in cultured fibroblasts. Fibroblasts treated with TGFβ-1 showed a pronounced up-regulation of BMP6, suggesting that stromal cells may be susceptible to BMP activation by cytokines with a proven role in the pathogenesis of PMF. We conclude that BMP family members may play an important role in the pathogenesis of myelofibrosis in PMF and are apparently induced by cytokines such as TGFβ-1. Primary myelofibrosis (PMF according to the latest proposals for a revised World Health Organization classification of the myeloproliferative disorders1Tefferi A Thiele J Orazi A Kvasnicka HM Barbui T Hanson CA Barosi G Verstovsek S Birgegard G Mesa R Reilly JT Gisslinger H Vannucchi AM Cervantes F Finazzi G Hoffman R Gilliland DG Bloomfield CD Vardiman JW Proposals and rationale for revision of the World Health Organization diagnostic criteria for polycythemia vera, essential thrombocythemia, and primary myelofibrosis: recommendations from an ad hoc international expert panel.Blood. 2007; 110: 1092-1097Crossref PubMed Scopus (759) Google Scholar) belongs to the group of Philadelphia chromosome-negative chronic myeloproliferative diseases. PMF may present in a prefibrotic, hypercellular phase2Thiele J Kvasnicka HM Prefibrotic chronic idiopathic myelofibrosis: a diagnostic enigma?.Acta Haematol. 2004; 111: 155-159Crossref PubMed Scopus (36) Google Scholar that progresses to bone marrow fibrosis during the course of the disease. Other prominent features comprise increased bone marrow angiogenesis, enhanced cellular trafficking of CD34+ progenitors, extramedullary hematopoiesis, and a variable risk for transformation into acute leukemia.3Tefferi A Pathogenesis of myelofibrosis with myeloid metaplasia.J Clin Oncol. 2005; 23: 8520-8530Crossref PubMed Scopus (218) Google Scholar, 4Xu M Bruno E Chao J Huang S Finazzi G Fruchtman SM Popat U Prchal JT Barosi G Hoffman R MPD Research Consortium Constitutive mobilization of CD34+ cells into the peripheral blood in idiopathic myelofibrosis may be due to the action of a number of proteases.Blood. 2005; 105: 4508-4515Crossref PubMed Scopus (96) Google Scholar In up to 60% of patients, constitutively activated Janus kinase (JAK2V617F) has been demonstrated to be an underlying molecular defect probably responsible for autonomous proliferation.5Campbell PJ Green AR The myeloproliferative disorders.N Engl J Med. 2006; 355: 2452-2466Crossref PubMed Scopus (518) Google Scholar A minor fraction (∼5%) of PMF may show a gain-of-function mutation in the thrombopoietin receptor myeloproliferative leukemia virus oncogene (MPLW515L/K).6Pardanani AD Levine RL Lasho T Pikman Y Mesa RA Wadleigh M Steensma DP Elliott MA Wolanskyj AP Hogan WJ McClure RF Litzow MR Gilliland DG Tefferi A MPL515 mutations in myeloproliferative and other myeloid disorders: a study of 1182 patients.Blood. 2006; 108: 3472-3476Crossref PubMed Scopus (832) Google ScholarThe definite mechanisms leading to myelofibrosis in PMF remain unclear, but it is well accepted that proliferation of fibroblasts represents a reactive and therefore nonclonal process.3Tefferi A Pathogenesis of myelofibrosis with myeloid metaplasia.J Clin Oncol. 2005; 23: 8520-8530Crossref PubMed Scopus (218) Google Scholar Induction and sustainment of bone marrow fibrosis are mediated by a complex network of cytokines with platelet-derived growth factor, basic fibroblast growth factor (bFGF), and transforming growth factor β-1 (TGFβ-1) being the major effector molecules in this process.7Le Bousse-Kerdilès MC Martyre MC Dual implication of fibrogenic cytokines in the pathogenesis of fibrosis and myeloproliferation in myeloid metaplasia with myelofibrosis.Ann Hematol. 1999; 78: 437-444Crossref PubMed Scopus (94) Google Scholar Besides the action of pro-fibrogenic factors with subsequent overproduction of extracellular matrix (ECM) components, the counteracting proteolytic environment, including collagenases/matrix metalloproteinases and their inhibitors, substantially contribute to the development of PMF.4Xu M Bruno E Chao J Huang S Finazzi G Fruchtman SM Popat U Prchal JT Barosi G Hoffman R MPD Research Consortium Constitutive mobilization of CD34+ cells into the peripheral blood in idiopathic myelofibrosis may be due to the action of a number of proteases.Blood. 2005; 105: 4508-4515Crossref PubMed Scopus (96) Google Scholar, 8Bock O Neuse J Hussein K Brakensiek K Buesche G Buhr T Wiese B Kreipe H Aberrant collagenase expression in chronic idiopathic myelofibrosis is related to the stage of disease but not to the JAK2 mutation status.Am J Pathol. 2006; 169: 471-481Abstract Full Text Full Text PDF PubMed Scopus (34) Google ScholarThe family of bone morphogenetic proteins (BMP) currently comprises 20 members9Hopkins DR Keles S Greenspan DS The bone morphogenetic protein 1/Tolloid-like metalloproteinases.Matrix Biol. 2007; 26: 508-523Crossref PubMed Scopus (198) Google Scholar. BMP1 to BMP7 are the most frequently investigated subtypes. The factor discovered first, BMP1, was initially extracted from bone lysates and was found to be capable of inducing ectopic bone formation.10Urist MR Bone: formation by autoinduction.Science. 1965; 150: 893-899Crossref PubMed Scopus (4449) Google Scholar, 11Urist MR Iwata H Ceccotti PL Dorfman RL Boyd SD McDowell RM Chien C Bone morphogenesis in implants of insoluble bone gelatin.Proc Natl Acad Sci USA. 1973; 70: 3511-3515Crossref PubMed Scopus (161) Google Scholar BMP1, unlike BMPs 2 to 7, is not a TGFβ-like protein but acts like a metalloproteinase. Hence, BMP1 is known to process a large set of ECM precursor molecules such as pro-collagen types I to III to mature fibrils. Furthermore, BMP1 cleaves the latent binding complex containing inactive TGFβ-1 in the ECM, thereby allowing matrix metalloproteinases such as matrix metalloproteinase-13 to fully activate TGFβ-1.12Ge G Greenspan DS BMP1 controls TGFbeta1 activation via cleavage of latent TGFbeta-binding protein.J Cell Biol. 2006; 175: 111-120Crossref PubMed Scopus (203) Google Scholar, 13D'Angelo M Billings PC Pacifici M Leboy PS Kirsch T Authentic matrix vesicles contain active metalloproteases (MMP): A role for matrix vesicle-associated MMP-13 in activation of transforming growth factor-beta.J Biol Chem. 2001; 276: 11347-11353Crossref PubMed Scopus (152) Google Scholar Interestingly, BMP1 also seems to mediate resistance of fibroblasts to apoptosis in vitro via processing of the proteoglycan Perlecan.14Laplante P Raymond MA Labelle A Abe J Iozzo RV Hébert MJ Perlecan proteolysis induces an alpha2beta1 integrin- and Src family kinase-dependent anti-apoptotic pathway in fibroblasts in the absence of focal adhesion kinase activation.J Biol Chem. 2006; 281: 30383-30392Crossref PubMed Scopus (62) Google Scholar In endothelial cells, BMP-1 overexpression was shown to be restricted to areas of tumor angiogenesis in vivo.15St Croix B Rago C Velculescu V Traverso G Romans KE Montgomery E Lal A Riggins GJ Lengauer C Vogelstein B Kinzler KW Genes expressed in human tumor endothelium.Science. 2000; 289: 1197-1202Crossref PubMed Scopus (1632) Google ScholarBMP family members other than BMP1 were shown to exert different effects on the development of fibrosis in solid organs and also tumorigenesis. For instance, BMP6 was a predominant mediator of fibrosis in experimental skin tumors,16Gitelman SE Kobrin MS Ye JQ Lopez AR Lee A Derynck R Recombinant Vgr-1/BMP-6-expressing tumors induce fibrosis and endochondral bone formation in vivo.J Cell Biol. 1994; 126: 1595-1609Crossref PubMed Scopus (146) Google Scholar whereas BMP7 was shown to protect against glomerular fibrosis and collagen accumulation in an animal model of diabetic nephropathy and in experimental cardiac fibrosis.17Wang S de Caestecker M Kopp J Mitu G Lapage J Hirschberg R Renal bone morphogenetic protein-7 protects against diabetic nephropathy.J Am Soc Nephrol. 2006; 17: 2504-2512Crossref PubMed Scopus (148) Google Scholar, 18Zeisberg EM Tarnavski O Zeisberg M Dorfman AL McMullen JR Gustafsson E Chandraker A Yuan X Pu WT Roberts AB Neilson EG Sayegh MH Izumo S Kalluri R Endothelial-to-mesenchymal transition contributes to cardiac fibrosis.Nat Med. 2007; 13: 952-961Crossref PubMed Scopus (1591) Google Scholar Interestingly, in a recent mouse model showing deficiency in GATA-1 expression (GATA-1low), members of the BMP family were shown to be essentially involved in early osteosclerosis.19Garimella R Kacena MA Tague SE Wang J Horowitz MC Anderson HC Expression of bone morphogenetic proteins and their receptors in the bone marrow megakaryocytes of GATA-1(low) mice: a possible role in osteosclerosis.J Histochem Cytochem. 2007; 55: 745-752Crossref PubMed Scopus (26) Google ScholarSite-directed mutagenesis in vitro provided evidence that a point mutation in human BMP1 (E483K) abolished the ability of BMP1 to cleave pro-collagen type I.20Hartigan N Garrigue-Antar L Kadler KE Bone morphogenetic protein-1 (BMP-1): identification of the minimal domain structure for procollagen C-proteinase activity.J Biol Chem. 2003; 278: 18045-18049Crossref PubMed Scopus (62) Google Scholar The entire CUB II domain was lastly identified to be essential for proteinase activity of BMP1.In summary, members of the BMP family seem to be involved in ECM synthesis and formation, homeostasis of fibroblasts, and angiogenesis rendering them attractive target molecules that contribute to myelofibrosis and angiogenesis, in PMF. To test the hypothesis of a relevant role of BMPs in PMF pathogenesis, we 1) analyzed BMP expression in fibroblasts treated with TGFβ-1 and bFGF in vitro, and 2) consecutively investigated PMF bone marrow samples for BMP expression in correlation with the stage of disease, ie, grade of myelofibrosis. An explorative mutation screening was performed to uncover a potential mutation in the CUB II domain of BMP1. To obtain a much closer insight into dynamics, we monitored BMP1 and BMP6 expression in PMF during the course of disease.Materials and MethodsBone Marrow Study GroupFormalin-fixed and paraffin-embedded bone marrow trephines with PMF diagnosed according to the World Health Organization criteria were retrieved from the bone marrow registry of the Institute of Pathology, Hannover Medical School. Bone marrow trephines were routinely fixed in phosphate-buffered formalin (pH 7.4) for 24 hours. The decalcification step was performed in an EDTA-based solution (pH 7.5) for up to 48 hours or by application of an ultrasound bath allowing a shortened processing period of less than 24 hours. The study group (n = 95) comprised hypercellular, prefibrotic PMF (n = 27), advanced PMF with manifest myelofibrosis (n = 43), and 25 control cases showing normal hematopoiesis or a mild reactive hyperplasia of the megakaryocytic lineage. According to the World Health Organization classification in close agreement with clinical data, patients' bone marrow trephines were diagnosed as PMF in the years 2000 to 2005. No prior history of PMF or a related chronic myeloproliferative disease was recorded in the study group. PMF cases were re-evaluated and subdivided into two groups depending on the degree of myelofibrosis (mf) after silver impregnation (Gomori) as described previously.21Thiele J Kvasnicka HM Facchetti F Franco V van der Walt J Orazi A European consensus on grading bone marrow fibrosis and assessment of cellularity.Haematologica. 2005; 90: 1128-1132PubMed Google Scholar, 22Buhr T Buesche G Choritz H Länger F Kreipe H Evolution of myelofibrosis in chronic idiopathic myelofibrosis as evidenced in sequential bone marrow biopsy specimens.Am J Clin Pathol. 2003; 119: 152-158Crossref PubMed Scopus (71) Google Scholar In brief, hypercellular PMF cases showing no deposition of fibers were graded as mf 0, and a mild increase of reticulin fibers (collagen type III) led to grade mf 1. Advanced PMF showing a manifest myelofibrosis with extended collagen deposits (type I + III) and intrasinusoidal hematopoiesis were graded as mf 2. In addition, demonstrable osteosclerosis and bone apposition were classified as grade mf 3. For a summary of patients' clinical data, see Table 1.Table 1Clinical Parameters, Including Frequency and Mutant T Allele Burden of JAK2V617F MutationsPMF mf 0/1 (n = 27)PMF mf 2/3 (n = 43)Control (n = 25)JAK2wt (n = 12)JAK2V617F (n = 15)JAK2wt (n = 21)JAK2V617F (n = 22)JAK2wt (n = 25)Mutant T allele burden (%)—34.0 (14.0 to 86.0)—50.0 (6.0 to 94.0)—Erythrocytes (106/μl)3.6 (2.2 to 5.3)4.5 (4.2 to 6.6)3.4 (2.7 to 4.0)4.0 (2.7 to 8.1)4.9 (3.7 to 5.7)Hemoglobin (g/dl)11.3 (9.6 to 14.2)14.1 (10.3 to 16.6)8.6 (6.3 to 10.8)10.8 (3.0 to 16.8)14.8 (9.4 to 16.6)Hematocrit (%)34 (30 to 49)41 (32 to 52)30 (26 to 40)33 (26 to 56)45 (31 to 50)Leukocytes (103/μl)8.2 (5.0 to 31.8)11.0 (2.9 to 22.9)9.5 (3.1 to 87.5)10.8 (3.8 to 48.1)6.2 (2.8 to 15.5)Thrombocytes (103/μl)696 (530 to 1919)711 (430 to 1340)343 (16 to 1081)257 (84 to 1415)212 (22 to 639)Age (years)67 (37 to 79)64 (34 to 81)62 (49 to 83)70 (50 to 83)59 (11 to 82)Sex (m/f)8/44/1115/612/1016/9No MPLW515L mutation was detected in the PMF group. The median values are displayed (range). wt, wild type. Open table in a new tab Sequential bone marrow trephines in another nine patients with PMF were investigated for individual dynamics of BMP1 and BMP6 mRNA expression during the course of the disease. Note that these nine individual patients are not part of the PMF group summarized in Table 1.Quantitative Genotyping for a Potential Underlying JAK2V617F and MPLW515L Mutation by PyrosequencingWe applied pyrosequencing assays that allow the quantification of mutant JAK2V617F or MPLW515L alleles in bone marrow cells.8Bock O Neuse J Hussein K Brakensiek K Buesche G Buhr T Wiese B Kreipe H Aberrant collagenase expression in chronic idiopathic myelofibrosis is related to the stage of disease but not to the JAK2 mutation status.Am J Pathol. 2006; 169: 471-481Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar Amplicons covering the hotspot point mutations (JAK2G1849T and MPLG1544T, respectively) were amplified by PCR from 25 ng of genomic DNA extracted by means of the Qiagen DNeasy kit (Qiagen, Hilden, Germany) using primers JAK2 forward, 5′-TATGATGAGCAAGCTTTCTCACAAG-3′; JAK2 reverse, 5′-AGAAAGGCATTAGAAAGCCTGTAGTT-3′ (GenBank accession no. AL161450) generating a 102-bp product; MPL forward, 5′-ATCTCCTTGGTGACCGCTCTG-3′; and MPL reverse, 5′-TGGTCCACCGCCAGTCTG-3′ (GenBank accession no. U68161) generating a 130-bp product with an additional 5′-biotin tag on respective reverse primers in a final volume of 50 μl. Pyrosequencing was performed as previously described.23Hussein K Brakensiek K Buesche G Buhr T Wiese B Kreipe H Bock O Different involvement of the megakaryocytic lineage by the JAK2 (V617F) mutation in Polycythemia vera, essential thrombocythemia and chronic idiopathic myelofibrosis.Ann Hematol. 2007; 86: 245-253Crossref PubMed Scopus (18) Google Scholar In brief, single-stranded PCR products were prepared by streptavidin Sepharose beat sorting and JAK2 and MPL specific sequencing (S) primers (JAK2-S 5′-GGTTTTAAATTATGGAGTATGT-3′, nucleotides 55039 to 55060 in GenBank accession no. AL161450, MPL-S 5′-GCCTGCTGCTGCTGAGGT-3′, nucleotides 1085 to 1102 in GenBank accession no. U68161) were applied to perform sequencing analysis and subsequent allele quantification in a PSQ 96MA instrument by using the SNP software (Biotage, Uppsala, Sweden). As described previously,8Bock O Neuse J Hussein K Brakensiek K Buesche G Buhr T Wiese B Kreipe H Aberrant collagenase expression in chronic idiopathic myelofibrosis is related to the stage of disease but not to the JAK2 mutation status.Am J Pathol. 2006; 169: 471-481Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar samples were scored as heterozygous for JAK2V617F if the percentage of mutant T alleles exceeded 5%. Homozygosity for JAK2V617F was considered when allele burden exceeded 50.0%.Samples under investigation were accompanied by positive controls (JAK2V617F cell lines HEL and SET-2; PMF patient with MPLW515L as evidenced by direct sequencing) and a negative control (JAK2wild-type/MPLwild-type cell line HL-60).Frequencies of potential JAK2V617F and MPLW515L mutations are summarized in Table 1.In Vitro Expression of BMPs by Human FibroblastsPrimary human fibroblasts (cell line F-18, kindly provided by Dr. Miriam Wittmann, Department of Dermatology and Allergology, Hannover Medical School, and cell line M15D24Schönermark MP Bock O Buechner A Steinmeier R Benbow U Lenarz T Quantification of tumor cell invasion using confocal laser scan microscopy.Nat Med. 1997; 3: 1167-1171Crossref PubMed Scopus (15) Google Scholar) were grown as monolayer cultures in RPMI containing 10% fetal calf serum and antibiotics until subconfluence was reached. Cells were then cultured for 24 hours in RPMI 1640 containing 0.5% fetal calf serum and antibiotics in the presence of recombinant bFGF (2 ng/ml, 2 μg/ml stock prepared in 4 mmol/L HCl; 234-FSE; R&D Systems, Minneapolis, MN) and recombinant TGFβ-1 (2 ng/ml, 2 μg/ml stock prepared in 4 mmol/L HCl; 240-B; R&D Systems). Controls were cultured in parallel by using solely the vehicle HCl in RPMI 1640 containing 0.5% fetal calf serum and antibiotics. In vitro experiments were performed in duplicate.Quantitative Real-Time PCRTotal RNA was extracted from cell cultures by using the TRIzol reagent (Invitrogen, Karlsruhe, Germany). As we previously described,25Bock O Kreipe H Lehmann U One-step extraction of RNA from archival biopsies.Anal Biochem. 2001; 295: 116-117Crossref PubMed Scopus (35) Google Scholar total RNA was extracted from total formalin-fixed and paraffin-embedded bone marrow cells after guanidinium isothiocyanate/Proteinase K-based digestion and conventional organic extraction using phenol/chloroform. Total RNA (1 μg), pretreated with RNase free (Rnase-) DNase (1 U/μg RNA; RQ1; Promega, Madison, WI), was transcribed into the complementary DNA using 500 ng of random hexamers (Amersham Pharmacia, Piscataway, NJ) and 200 U of SuperScript II Rnase-Reverse Transcriptase (Invitrogen) in a volume of 20 μl following the manufacturer's protocol. Negative controls were performed by pipetting water instead of reverse transcriptase. Real-time RT-PCR was performed on an ABI PRISM 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA).After determination of PCR efficiency and demonstration of linearity of PCR amplification for target and housekeeping genes, a relative quantification approach was performed using the ΔΔCT− method as described previously.8Bock O Neuse J Hussein K Brakensiek K Buesche G Buhr T Wiese B Kreipe H Aberrant collagenase expression in chronic idiopathic myelofibrosis is related to the stage of disease but not to the JAK2 mutation status.Am J Pathol. 2006; 169: 471-481Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 26Livak KJ Schmittgen TD Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT Method.Methods. 2001; 25: 402-408Crossref PubMed Scopus (119422) Google Scholar All samples were measured in duplicate. Sequences of PCR primers and TaqMan probes used in this study are listed in Table 2.Table 2Primer and Probe Sequences Used throughout This StudyPrimer and probesSequence or published referenceGenBank accession number, amplicon size (bp)BMP1 forward5′-CAGTCCTTTGAGATTGAGCGC-3′BMP1 reverse5′-TGCTGCTCTCACTGTGCCC-3′BMP1 probe5′-ACGACAGCTGTGCCTACGACTATCTGGAGGT-3′NM 001199, 79BMP2 forward5′-CAACACTGTCGCAGCTTC-3′BMP2 reverse5′-GAAGAATCTCCGGGTTGTTTTC-3′BMP2 probe5′-CCATGAAGAATCTTTGGAAGAACTACCAGAAACGAGT-3′NM001200.2, 82BMP4 forward5′-ACTGGTCTTGAGTATCCTGAGCGC-3′BMP4 reverse5′-AAGCAGAGTTTTCACTGGTCCCT-3′BMP4 probe5′-TTCCACCACGAAGAACATCTGGAGAACATCC-3′NM001202.1, 109BMP6 forward5′-GGAAGCATGAGCTGTATGTGAGTTT-3′BMP6 reverse5′-AGTAATTGGCAGCATAGCATAGCCCTTG-3′BMP6 probe5′-AAGACCTGGGATGGCAGGACTGGATCA-3′NM001218.2, 84BMP7 forward5′-CAAGAACCAGGAAGCCCTGC-3′BMP7 reverse5′-TTCTTACAGGCCTGCCTCTGG-3′BMP7 probe5′-ATGGCCAACGTGGCAGAGAACAGCA-3′NM001719, 75BMP-R2 forward5′-CTTTACTGAGAATTTTCCACCTCCTG-3′BMP-R2 reverse5′-GCCAAAGCAATGATTATTGTCTCATC-3′BMP-R2 probe5′-CACAACACCACTCAGTCCACCTCATTCATTTAAC-3′NM001204.5, 90PLOD2 forward5′-ATGGACACAGGATAATGGCTGC-3′PLOD2 reverse5′-CACCTATTGATACGTTTGGATGGAC-3′PLOD2 probe5′-CTCTTTGTGAATTCGATACAGTCGACTTGTCTGC-3′NM 182943, 89BMP1-CUBII forward5′-ACTGATGAAGCCTCGACCCC-3′BMP1-CUBII reverse5′-AGAGTAACAGAGGAGGCACCTTTG-3′NT023666, 237COL1Ref.8Bock O Neuse J Hussein K Brakensiek K Buesche G Buhr T Wiese B Kreipe H Aberrant collagenase expression in chronic idiopathic myelofibrosis is related to the stage of disease but not to the JAK2 mutation status.Am J Pathol. 2006; 169: 471-481Abstract Full Text Full Text PDF PubMed Scopus (34) Google ScholarNM 000088, 90β-GUSRef.8Bock O Neuse J Hussein K Brakensiek K Buesche G Buhr T Wiese B Kreipe H Aberrant collagenase expression in chronic idiopathic myelofibrosis is related to the stage of disease but not to the JAK2 mutation status.Am J Pathol. 2006; 169: 471-481Abstract Full Text Full Text PDF PubMed Scopus (34) Google ScholarNM 000181, 81HSP-70.1Ref.8Bock O Neuse J Hussein K Brakensiek K Buesche G Buhr T Wiese B Kreipe H Aberrant collagenase expression in chronic idiopathic myelofibrosis is related to the stage of disease but not to the JAK2 mutation status.Am J Pathol. 2006; 169: 471-481Abstract Full Text Full Text PDF PubMed Scopus (34) Google ScholarNM 005345, 62Probes were labeled with 5′-FAM and 3′-TAMRA. Open table in a new tab Explorative Screening for a Potential Point Mutation (E483K) in the CUB II Domain of BMP1 in PMFA 237-bp DNA fragment was amplified covering the potential hotspot mutation G1476A (GenBank accession no. NM001199) in the CUB II domain of BMP1. Amplification of 25 ng DNA derived from total bone marrow cells of advanced PMF (n = 22) and controls (n = 3) was performed in a GeneAmp PCR System 2700 (Applied Biosystems) followed by direct sequencing in an ABI 310 Genetic Analyzer (Applied Biosystems). Primer sequences are listed in Table 2.ImmunohistochemistryImmunohistochemistry for BMP1 in PMF (mf 0/1, n = 10; mf 2/3, n = 15) and control bone marrows (n = 10) was performed on bone marrow slides with a goat anti-human BMP1/PCP antibody (AF1927; R&D Systems). This antibody recognizes the amino acid sequence 121 to 730 of the mature BMP1 protein. After pretreatment in a pressure cooker at 125°C in citrate buffer (10 mmol/L, pH 6.0) for 3 minutes, the primary antibody was applied in a dilution of 1:50 before incubation for 1 hour at room temperature. Visualization was performed with the DAB Zytomed kit (DAB057; Zytomed Systems, Berlin, Germany) according to the manufacturer's instructions and by counterstaining with hematoxylin. The protocol was strictly accompanied by a positive control (kidney tissue) and negative controls (samples were processed by omission of primary antibodies).Statistics and GraphicsTo analyze differences of mRNA expression in prefibrotic PMF, advanced PMF, and non-neoplastic hematopoiesis, nonparametric Kruskal-Wallis tests were performed followed by Dunns post test for pairwise group differences. For potential correlation of JAK2 status (allele burden) and BMP expression, unpaired t-tests and one-way analysis of variance tests were performed. P values ≤0.05 were considered as statistically significant. Graphics were designed using GraphPadPrism, Version 5.0 (GraphPad Software, San Diego, CA) and SigmaPlot, Version 8.0 (SPSS, Inc., Chicago, IL).ResultsTGFβ-1 and bFGF Induce Overexpression of BMPs in Cultured FibroblastsThe in vitro treatment of fibroblasts (cell lines M15D and F-18) with TGFβ-1 showed a prominent induction of target gene expression for all BMPs, including BMP-R2, except for BMP7. In contrast to untreated fibroblasts, BMP7 was undetectable by qualitative and quantitative PCR approaches. Fibroblasts cultured in the presence of bFGF showed a different response with restricted induction of BMP6 in M15D and F-18 but not other BMPs. Increased expression of collagen-type 1 (COL1) and procollagen-lysine 2-oxoglutarate 5-dioxygenase 2 (PLOD2) served as markers for sufficient induction of collagen synthesis by TGFβ-1. No induction of COL1 and PLOD2 was observed after stimulation by bFGF (Figure 1; Table 3).Table 3Relative Levels of BMP Expression in Fibroblasts (M15D, F-18) Cultured in the Presence of TGFβ-1 and bFGFM15DTGF-β1 [median (range)]bFGF [median (range)]F-18TGF-β1 [median (range)]bFGF [median (range)]BMP11.8 (1.6 to 1.9)0.7 (0.6 to 0.8)BMP 12.3 (1.9 to 2.7)0.6 (0.5 to 0.6)BMP614.4 (13.9 to 14.9)4.8 (1.9 to 7.7)BMP 612.5 (5.3 to 19.7)1.8 (0.2 to 3.5)BMP-R22.5 (2.4 to 2.6)1.2 (1.0 to 1.4)BMP to R23.0 (2.9 to 3.1)0.3 (0.2 to 0.5)PLOD26.2 (2.5 to 9.9)0.5 (0.1 to 1.0)PLOD 26.7 (4.4 to 8.9)1.0 (0.9 to 1.0)COL12.0 (2.0 to 2.1)0.3 (0.2 to 0.3)COL 12.3 (1.9 to 2.6)0.2 (0.2 to 0.3) Open table in a new tab PLOD2 Is Overexpressed in the Advanced Stages of PMF (mf 2/3)We investigated a series of PMF (mf 0/1, n = 10; mf 2/3, n = 11) and control cases (n = 10) for PLOD2 mRNA expression to reproduce the data of PLOD2 mRNA induction by TGFβ-1 during in vitro culture of fibroblasts. We found a significantly higher PLOD2 mRNA expression level in PMF mf 2/3 (median, 3.0; range, 0.3 to 6.9) compared with controls (median, 1.0; range, 0.5 to 2.6, P = 0.01), and a trend toward a significant difference when compared with PMF mf 0/1 (median, 1.1; range, 0.4 to 5.0; P = 0.05). PMF mf 0/1 did not significantly differ from controls (P = 0.44).BMP1, -6, -7, and BMP-R2 mRNA Expression in Different Stages of PMFAdvanced PMF stages showed a significantly increased BMP1 mRNA expression of up to fivefold compared with the hypercellular, prefibrotic stages (P < 0.01) and non-neoplastic hematopoiesis (P < 0.001). The prefibrotic stage and advanced stages of PMF significantly overexpressed BMP6 compared with control bone marrows (P < 0.01 and 0.001, respectively). BMP7 was increased in advanced stages compared with the prefibrotic stage and controls (P < 0.05). BMP-R2 was overexpressed in advanced PMF compared with controls (P < 0.05). BMP2 expression showed no difference between the groups under investigation (Figure 2).Figure 2Point plot illustration of BMP mRNA expression in bone marrow cells of hypercellular, prefibrotic PMF (PMF mf 0/1), advanced PMF (PMF mf 2/3), and controls. Horizontal bars represent median values of expression. P values denote statistical relevance. Note that BMP mRNA expression in control hematopoiesis was arbitrarily set to 1. A comprehensive illustration of expression levels and potential correlation to grade of myelofibrosis and underlying molecular defects is displayed in Table 4, Table 5.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Of note, only 20% of the cases showed an amplifiable BMP4 mRNA in the qualitative RT-PCR approach. Therefore, no further analysis of BMP4 by quantitative approaches was undertaken. A comprehensive compilation of BMP expression levels is shown in Table 4.Table 4Relative Levels of BMP Expression by Bone Marrow C
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