Analysis of TGFβ3 gene expression and protein levels in human bone and serum

Summary:

Recent data indicate that TGFβ3, one member of the TGFβ-isoforms, has an important role in bone remodeling. Up to date little is known about the expression and regulation of TGFβ3 in man. We established a highly specific ELISA for quantitative measurement of TGFβ3 in bone and blood samples and a RT-PCR in combination with HPLC for detection and quantification of TGFβ3 mRNA in 89 human bone samples. Levels of TGFβ3 protein ranged between 30 and 66 pg/mg bone (mean 36,6 ± 1,03 pg/mg) and between 30 and 1910 pg/ml in serum (mean 128,9 ± 38.9 pg/ml). TGFβ3 mRNA expression as well as protein levels in serum and in bone declined age dependently. No specific load- or site-specific distribution of TGFβ3 mRNA expression or protein content was detected at different sites indicating an absence of mechanical regulation. Protein levels of TGFβ3 in serum correlated with TGFβ3 mRNA expression in bone (p = 0.0027; r = 0.49). By contrast, TGFβ3 protein levels stored in the bone matrix were not related to TGFβ3 mRNA reflecting the long term process of TGFβ3 deposition during bone remodeling. Notably TGFβ3 serum levels were highly correlated with IGF-I and osteocalcin levels in serum. We conclude that TGFβ3 in man circulates in significant amounts which appears to be representative for TGFβ3 expression in bone tissue and may be in part derived from bone. The high correlation of TGFβ3 with IGF-I suggests parallel systemic principles of regulation.

Key words:

HPLC - TGFβ3-ELISA - aging - quantative-PCR - bone biopsy

References

• 1 Bismar H, Diel I, Ziegler R, Pfeilschifter J. Increased cytokine secretion by human bone marrow cells after menopause or discontinuation of estrogen replacement.  J Clin Endocrinol Metab. 80 3351-3355 1995; 
• 2 Blum W F, Ranke M B, Bierich J R. A specific radioimmunoassay for insulin-like growth factor II: the interference of IGF binding proteins can be blocked by excess IGF-I.  Acta Endocrinol (Copenh). 118 374-380 1988; 
• 3 Cheifetz S, Hernandez H, Laiho M, Ten Dijke P, Iwata K K, Massague J. Distinct transforming growth factor-beta (TGF-beta) receptor subsets as determinants of cellular responsiveness to three TGF-beta isoforms.  J Biol Chem. 265 20533-20538 1990; 
• 4 Cheifetz S, Weatherbee J A, Tsang M L, Anderson J K, Mole J E, Lucas R, Massague J. The transforming growth factor-beta system, a complex pattern of cross-reactive ligands and receptors.  Cell. 48 409-415 1987; 
• 5 Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.  Anal Biochem. 162 156-159 1987; 
• 6 Danielpour D, Roberts A B. Specific and sensitive quantitation of transforming growth factor beta 3 by sandwich enzyme-linked immunosorbent assay.  J Immunol Methods. 180 265-272 1995; 
• 7 Denhez F, Lafyatis R, Kondaiah P, Roberts A B, Sporn M B. Cloning by polymerase chain reaction of a new mouse TGF-beta, mTGF-beta 3.  Growth Factors. 3 139-146 1990; 
• 8 Dequeker J, Mohan S, Finkelman R D, Aerssens J, Baylink D J. Generalized osteoarthritis associated with increased insulin-like growth factor types I and II and transforming growth factor beta in cortical bone from the iliac crest. Possible mechanism of increased bone density and protection against osteoporosis.  Arthritis Rheum. 36 1702-1708 1993; 
• 9 Derynck R, Lindquist P B, Lee A, Wen D, Tamm J, Graycar J L, Rhee L, Mason A J, Miller D A, Coffey R J. et al . A new type of transforming growth factor-beta, TGF-beta 3.  Embo J. 7 3737-3743 1988; 
• 10 Edwards D R, Leco K J, Beaudry P P, Atadja P W, Veillette C, Riabowol K T. Differential effects of transforming growth factor-beta 1 on the expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in young and old human fibroblasts.  Exp Gerontol. 31 207-223 1996; 
• 11 Frazer A, Seid J M, Hart K A, Bentley H, Bunning R A, Russell R G. Detection of mRNA for the transforming growth factor beta family in human articular chondrocytes by the polymerase chain reaction.  Biochem Biophys Res Commun. 180 602-608 1991; 
• 12 Gatherer D, Ten Dijke P, Baird D T, Akhurst R J. Expression of TGF-beta isoforms during first trimester human embryogenesis.  Development. 110 445-460 1990; 
• 13 Grainger D J, Percival J, Chiano M, Spector T D. The role of serum TGF-beta isoforms as potential markers of osteoporosis.  Osteoporos Int. 9 398-404 1999; 
• 14 Henninger H P, Hoffmann R, Grewe M, Schulze-Specking A, Decker K. Purification and quantitative analysis of nucleic acids by anion-exchange high-performance liquid chromatography.  Biol Chem Hoppe Seyler. 374 625-634 1993; 
• 15 Horner A, Kemp P, Summers C, Bord S, Bishop N J, Kelsall A W, Coleman N, Compston J E. Expression and distribution of transforming growth factor-beta isoforms and their signaling receptors in growing human bone.  Bone. 23 95-102 1998; 
• 16 Jarvinen T L, Kannus P, Sievanen H, Jolma P, Heinonen A, Jarvinen M. Randomized controlled study of effects of sudden impact loading on rat femur.  J Bone Miner Res. 13 1475-1482 1998; 
• 17 Joyce M E, Jingushi S, Bolander M E. Transforming growth factor-beta in the regulation of fracture repair.  Orthop Clin North Am. 21 199-209 1990; 
• 18 Katz E D, Haff L A, Eksteen R. Rapid separation, quantitation and purification of products of polymerase chain reaction by liquid chromatography.  J Chromatogr. 512 433-444 1990; 
• 19 Klein-Nulend J, Roelofsen J, Sterck J G, Semeins C M, Burger E H. Mechanical loading stimulates the release of transforming growth factor-beta activity by cultured mouse calvariae and periosteal cells.  J Cell Physiol. 163 115-119 1995; 
• 20 Koike K, Urata Y, Koike M. Molecular cloning and characterization of human pyruvate dehydrogenase beta subunit gene.  Proc Natl Acad Sci USA. 87 5594-5597 1990; 
• 21 Lee C KKR, Weindruch R, Prolla T A. Gene expression profile of aging and its retardation by caloric restriction.  Science. 285 1390-1393 1999; 
• 22 Lotz M, Rosen F, McCabe G, Quach J, Blanco F, Dudler J, Solan J, Goding J, Seegmiller J E, Terkeltaub R. Interleukin 1 beta suppresses transforming growth factor-induced inorganic pyrophosphate (PPi) production and expression of the PPi-generating enzyme PC-1 in human chondrocytes.  Proc Natl Acad Sci USA. 92 10364-10368 1995; 
• 23 Massague J. The transforming growth factor-beta family.  Annu Rev Cell Biol. 6 597-641 1990; 
• 24 Millan F A, Denhez F, Kondaiah P, Akhurst R J. Embryonic gene expression patterns of TGF beta 1, beta 2 and beta 3 suggest different developmental functions in vivo.  Development. 111 131-143 1991; 
• 25 Miller D A, Lee A, Matsui Y, Chen E Y, Moses H L, Derynck R. Complementary DNA cloning of the murine transforming growth factor-beta 3 (TGF beta 3) precursor and the comparative expression of TGF beta 3 and TGF beta 1 messenger RNA in murine embryos and adult tissues.  Mol Endocrinol. 3 1926-1934 1989; 
• 26 Nakajima Y, Yamagishi T, Nakamura H, Markwald R R, Krug E L. An autocrine function for transforming growth factor beta 3 in the atrioventricular endocardial cushion tissue formation during chick heart development.  Ann NY Acad Sci. 857 272-275 1998; 
• 27 Nicolas V, Prewett A, Bettica P, Mohan S, Finkelman R D, Baylink D J, Farley J R. Age-related decreases in insulin-like growth factor-I and transforming growth factor-beta in femoral cortical bone from both men and women: implications for bone loss with aging [see comments].  J Clin Endocrinol Metab. 78 1011-1016 1994; 
• 28 Oursler M J, Cortese C, Keeting P, Anderson M A, Bonde S K, Riggs B L, Spelsberg T C. Modulation of transforming growth factor-beta production in normal human osteoblast-like cells by 17 beta-estradiol and parathyroid hormone.  Endocrinology. 129 3313-3320 1991; 
• 29 Overall C M. A microtechnique for dialysis of small volume solutions with quantitative recoveries.  Anal Biochem. 165 208-214 1987; 
• 30 Pelton R W, Saxena B, Jones M, Moses H L, Gold L I. Immunohistochemical localization of TGF beta 1, TGF beta 2, and TGF beta 3 in the mouse embryo: expression patterns suggest multiple roles during embryonic development.  J Cell Biol. 115 1091-1095 1991; 
• 31 Pfeilschifter J, Diel I, Scheppach B, Bretz A, Krempien R, Erdmann J, Schmid G, Reske N, Bismar H, Seck T, Krempien B, Ziegler R. Concentration of transforming growth factor beta in human bone tissue: relationship to age, menopause, bone turnover, and bone volume.  J Bone Miner Res. 13 716-730 1998; 
• 32 Pfeilschifter J, Erdmann J, Storch S, Ziegler R, Weinreb M. Changes in the concentration of insulin-like growth factor I and transforming growth factor beta1 in rat femoral bone during growth.  Calcif Tissue Int. 64 78-82 1999; 
• 33 Pfeilschifter J, Laukhuf F, Muller-Beckmann B, Blum W F, Pfister T, Ziegler R. Parathyroid hormone increases the concentration of insulin-like growth factor-I and transforming growth factor beta 1 in rat bone.  J Clin Invest. 96 767-774 1995; 
• 34 Pfeilschifter J, Scheidt-Nave C, Leidig-Bruckner G, Woitge H W, Blum W F, Wuster C, Haack D, Ziegler R. Relationship between circulating insulin-like growth factor components and sex hormones in a population-based sample of 50- to 80-year-old men and women.  J Clin Endocrinol Metab. 81 2534-2540 1996; 
• 35 Riggs B L, Melton L Jd. Evidence for two distinct syndromes of involutional osteoporosis.  Am J Med. 75 899-901 1983; 
• 36 Sarkissian M, Lafyatis R. Transforming growth factor-beta and platelet derived growth factor regulation of fibrillar fibronectin matrix formation by synovial fibroblasts.  J Rheumatol. 25 613-622 1998; 
• 37 Shipley G D, Tucker R F, Moses H L. Type beta transforming growth factor/growth inhibitor stimulates entry of monolayer cultures of AKR-2B cells into S phase after a prolonged prereplicative interval.  Proc Natl Acad Sci USA. 82 4147-4151 1985; 
• 38 Su S, Grover J, Roughley P J, DiBattista J A, Martel-Pelletier J, Pelletier J P, Zafarullah M. Expression of the tissue inhibitor of metalloproteinases (TIMP) gene family in normal and osteoarthritic joints.  Rheumatol Int. 18 183-191 1999; 
• 39 Tardif G, Pelletier J P, Dupuis M, Geng C, Cloutier J M, Martel-Pelletier J. Collagenase 3 production by human osteoarthritic chondrocytes in response to growth factors and cytokines is a function of the physiologic state of the cells.  Arthritis Rheum. 42 1147-1158 1999; 
• 40 Westerlind K C, Turner R T. The skeletal effects of spaceflight in growing rats: tissue-specific alterations in mRNA levels for TGF-beta.  J Bone Miner Res. 10 843-848 1995; 
• 41 Yang N N, Bryant H U, Hardikar S, Sato M, Galvin R J, Glasebrook A L, Termine J D. Estrogen and raloxifene stimulate transforming growth factor-beta 3 gene expression in rat bone: a potential mechanism for estrogen- or raloxifene-mediated bone maintenance.  Endocrinology. 137 2075-2084 1996; 
• 42 Zaman G, Suswillo R F, Cheng M Z, Tavares I A, Lanyon L E. Early responses to dynamic strain change and prostaglandins in bone-derived cells in culture.  J Bone Miner Res. 12 769-777 1997;