Fluid Shear of Low Magnitude Increases Growth and Expression of TGFβ1 and Adhesion Molecules in Human Bone Cells in Vitro

U. M. Liegibel; U. Sommer; B. Bundschuh; B. Schweizer; U. Hilscher; A. Lieder; P. Nawroth; C. Kasperk

doi:10.1055/s-2004-821014

Experimental and Clinical Endocrinology & Diabetes, Table of Contents

Exp Clin Endocrinol Diabetes 2004; 112(7): 356-363
DOI: 10.1055/s-2004-821014

Article

J. A. Barth Verlag in Georg Thieme Verlag KG Stuttgart · New York

Fluid Shear of Low Magnitude Increases Growth and Expression of TGFβ1 and Adhesion Molecules in Human Bone Cells in Vitro

U. M. Liegibel¹ , U. Sommer¹ , B. Bundschuh¹ , B. Schweizer² , U. Hilscher¹ , A. Lieder¹ , P. Nawroth¹ , C. Kasperk¹

¹Endocrinology and Metabolism, Department of Medicine, Ruprecht-Karls-University, Heidelberg, Germany
²Institute of Applied Mathematics, Ruprecht-Karls-University, Heidelberg, Germany

Abstract

Deformation of the bone matrix by mechanical strain causes fluid shifts within the osteocytic canaliculi which affect osteocytic cell metabolism. We applied low fluid shear (1 - 63 µPa for 10 - 48 h) to human osteoblastic cells (HOB) in vitro to study its impact on cell proliferation and differentiated functions. Proteins involved in translating the physical force into a cellular response were characterised. Low fluid shear stress stimulated proliferation of HOB 1.2-fold when stress was applied intermittently for 24 h. Shear stress also increased differentiated cellular properties such as alkaline phosphatase (ALP) activity (121 % of control), fibronectin (FN) and fibronectin receptor (FNR) expression (290 % and 200 %, respectively). Prostaglandin E₂ (PGE₂) and TGFβ1 release into the medium were significantly stimulated when shear stress was applied for 6 - 12 h and 24 - 48 h, respectively. TGFβ1 + 2 neutralising antibodies or the presence of indomethacine inhibited the mitogenic effect of fluid shear and reduced ALP activity to its control level. Furthermore, TGFβ treatment induced a dose-dependent increase in FN and FNR expression. Therefore, fluid shear stress of low magnitude (a) suffices to affect HOB metabolism and (b) regulates anchorage of HOB via FN and FNR by stimulating osteoblastic PGE₂ and TGFβ secretion.

Key words

Fluid shear stress - osteoblast - fibronectin - integrin - gravitation

Full Text

References

1 Adams D S. Mechanisms of cell shape change: the cytomechanics of cellular response to chemical environment and mechanical loading. J Cell Biol. 1992; 117 83-93
2 Bancroft G N, Sikavitsas V I, van den Dolder J, Sheffield T L, Ambrose C G, Jansen J A, Mikos A G. Fluid flow increases mineralized matrix deposition in 3 D perfusion culture of marrow stromal osteoblasts in a dose-dependent manner. Proc Natl Acad Sci USA. 2002; 99 12600-12605
3 Bergula A P, Huang W, Frangos J A. Femoral vein ligation increases bone mass in the hindlimb suspended rat. Bone. 1999; 24 171-177
4 Bikle D D, Halloran B P. The response of bone to unloading. Bone Miner Metab. 1999; 17 233-244
5 Carmeliet G, Bouillon R. The effect of microgravity on morphology and gene expression of osteoblasts in vitro. FASEB J. 1999; 13 (Suppl) 129-134
6 Carvalho R S, Scott J E, Suga D M, Yen E H. Stimulation of signal transduction pathways in osteoblasts by mechanical strain potentiated by parathyroid hormone. J Bone Miner Res. 1994; 9 999-1011
7 Chen Q, Kinch M S, Lin T H, Burridge K, Juliano R L. Integrin-mediated cell adhesion activates mitogen-activated protein kinases. J Biol Chem. 1994; 269 26602-26605
8 Cheng B, Zhao S, Luo J, Sprague E, Bonewald L F, Jiang J X. Expression of functional gap junctions and regulation by fluid flow in osteocyte-like MLO-Y4 cells. J Bone Miner Res. 2001; 16 249-259
9 Damien E, Price J S, Lanyon L E. Mechanical strain stimulates osteoblast proliferation through the estrogen receptor in males as well as females. J Bone Miner Res. 2000; 15 2169-2176
10 Dehority W, Halloran B P, Bikle D D, Curren T, Kostenuik P J, Wronski T J, Shen Y, Rabkin B, Bouraoui A, Morey-Holton E. Bone and hormonal changes induced by skeletal unloading in the mature male rat. Am J Physiol. 1999; 276 E62-69
11 Fermor B, Gundle M, Emerton M, Pocock A, Murray D. Primary human osteoblast proliferation and prostaglandin E2 release in response to mechanical strain in vitro. Bone. 1998; 22 637-643
12 Fukuda S, Yasu T, Predescu D N, Schmid-Schönbein G W. Mechanisms for regulation of fluid shear stress response in circulating leukocytes. Circ Res. 2000; 86 e13-e18
13 Gadeleta S J, Boskey A L, Paschalis E, Carlson C, Menschik F, Baldini T, Peterson M, Rimnac C M. A physical, chemical, and mechanical study of lumbar vertebrae from normal, ovariectomized, and nandrolone decanoate treated cynomolgus monkeys. Bone. 2000; 27 541-550
14 Gazit D, Zilberman Y, Ebner R, Kahn A. Bone loss (osteopenia) in old mice results from diminished activity and availability of TGF-beta. J Cell Biochem. 1998; 70 478-488
15 Giancotti F G, Ruoslahti E. Integrin signaling. Science. 1999; 285 1028-1032
16 Gronthos S, Simmons P J, Graves S E, Robey P G. Integrin-mediated interactions between human bone marrow stromal precursor cells and the extracellular matrix. Bone. 2001; 28 174-181
17 Gronthos S, Stewart K, Graves S E, Hay S, Simmons P J. Integrin expression and function on human osteoblast-like cells. J Bone Miner Res. 1997; 12 1189-1197
18 Halloran B P, Bikle D D, Harris J, Tanner S, Curren T, Morey-Holton E. Regional responsiveness of the tibia to intermittent administration of parathyroid hormone as affected by skeletal unloading. J Bone Miner Res. 1997; 12 1068-1074
19 Hatton J P, Pooran M, Li C-F, Luzzio C, Hughes-Fulford M. A short pulse of mechanical force induces gene expression and growth in MC3T3-E1 osteoblasts via an ERK 1/2 pathway. J Bone Miner Res. 2003; 18 58-66
20 Howe A, Aplin A E, Alahari S K, Juliano R L. Integrin signaling and cell growth control. Curr Opinion Cell Biol. 1998; 10 220-231
21 Ignotz R A, Massague J. Cell adhesion protein receptors as targets for transforming growth factor-β action. Cell. 1987; 51 189-197
22 Jee W SS, Akamine T, Ke H Z, Li X J, Tang L Y, Zeng Q Q. Prostaglandin prevents disuse-induced cortical bone loss. Bone. 1992; 13 153-159
23 Jessop H L, Rawlinson S CF, Pitsillides A A, Lanyon L E. Mechanical strain and fluid movement both activate extracellular regulated kinase (ERK) in osteoblast-like cells but via different signalling pathways. Bone. 2002; 31 186-194
24 Jiang G L, White C R, Stevens H Y, Frangos J A. Temporal gradients in shear stimulate osteoblastic proliferation via ERK 1/2 and retinoblastoma protein. Am J Physiol Endocrinol Metab. 2002; 283 E383-E389
25 Kapur S, Baylink D J, Lau K HW. Fluid flow shear stress stimulates human osteoblast proliferation and differentiation through multiple interacting and competing signal transduction pathways. Bone. 2003; 32 241-251
26 Kaspar D, Seidl W, Neidlinger-Wilke C, Ignatius A, Claes L. Dynamic cell stretching increases human osteoblast proliferation and CICP synthesis but decreases osteocalcin synthesis and alkaline phosphatase activity. J Biomech. 2000; 33 45-51
27 Kasperk C H, Wergedal J E, Strong D D, Farley J R, Wangerin K, Gropp H, Ziegler R, Baylink D J. Human bone cell phenotypes differ depending on their skeletal site of origin. J Clin Endocrinol Metab. 1995; 80 2511-2517
28 Kasra M, Grynpas M D. The effects of androgens on the mechanical properties of primate bone. Bone. 1995; 17 265-270
29 Kassem M, Kveiborg M, Eriksen E F. Production and action of transforming growth factor-beta in human osteoblast cultures: dependence on cell differentiation and modulation by calcitriol. Eur J Clin Invest. 2000; 30 429-437
30 Klein-Nulend J, Burger E H, Semeins C M, Raisz L G, Pilbeam C C. Pulsating fluid flow stimulates prostaglandin release and inducible prostaglandin G/H synthase mRNA expression in primary mouse bone cells. J Bone Miner Res. 1997; 12 45-51
31 Klein-Nulend J, Semeins C M, Burger E H. Prostaglandin mediated modulation of transforming growth factor β metabolism in primary mouse osteoblastic cells in vitro. J Cellular Physiol. 1996; 168 1-7
32 Kontulainen S, Kannus P, Haapasalo H, Sievänen H, Pasanen M, Heinonen A, Oja P, Vuori I. Good maintenance of exercise induced bone gain with decreased training of female tennis and squash players: a prospective 5 year follow-up of young and old starters and controls. J Bone Miner Res. 2001; 16 195-205
33 Kurata K, Uemura T, Nemoto A, Tateishi T, Murakami T, Higaki H, Miura H, Iwamoto Y. Mechanical strain effect on bone-resorbing activity and messenger RNA expressions of marker enzymes in isolated osteoclast culture. J Bone Miner Res. 2001; 16 722-730
34 Leblanc A D, Schneider V S, Evans H J, Engelbretson D A, Krebs J M. Bone mineral loss and recovery after 17 weeks of bed rest. J Bone Miner Res. 1990; 5 843-850
35 Liegibel U M, Sommer U, Tomakidi P, Hilscher U, van den Heuvel L, Pirzer R, Hillmeier J, Nawroth P, Kasperk C. Concerted action of androgens and mechanical strain shifts bone metabolism from high turnover into an osteoanabolic mode. J Exp Med. 2002; 196 1387-1392
36 Ma Y, Jee W SS, Yuan Z, Wei W, Chen H, Pun S, Liang H, Lin C. Parathyroid hormone and mechanical usage have a synergistic effect in rat tibial diaphyseal cortical bone. J Bone Miner Res. 1999; 14 439-448
37 McAllister T N, Frangos J A. Steady and transient fluid shear stress stimulate NO release in osteoblasts through distinct biochemical pathways. J Bone Miner Res. 1999; 14 930-936
38 Moazzam F, DeLano F A, Zweifach B W, Schmid-Schönbein G W. The leukocyte response to fluid stress. Proc Natl Acad Sci USA. 1997; 94 5338-5343
39 Oktay M, Wary K K, Dans M, Birge R B, Giancotti K C. Integrin-mediated activation of focal adhesion kinase is required for signaling to Jun NH₂-terminal kinase and progression through the G1 phase of the cell cycle. J Cell Biol. 1999; 145 1461-1469
40 Owen I, Burr D, Turner C H, Qiu J, Onyia J, Duncan R L. Mechanotransduction in bone: Osteoblasts are more responsive to fluid forces than mechanical strain. Am J Physiol. 1997; 42 C810-C815
41 Pilbeam C, Rao Y, Voznesensky O, Kawaguchi H, Alander C, Raisz L, Herschman H. Transforming growth factor β1 regulation of prostaglandin G/H synthase 2 expression in osteoblastic MC3T3-E1 cells. Endocrinology. 1997; 138 4672-4682
42 Rawlinson S CF, Wheeler-Jones C PD, Lanyon L E. Arachidonic acid for loading induced prostacyclin and prostaglandin E2 release from osteoblasts and osteocytes is derived from the activities of different forms of phospholipase A2. Bone. 2000; 27 241-247
43 Reich K M, McAllister T N, Gudi S, Frangos J A. Activation of G proteins mediates flow-induced prostaglandin E₂ production in osteoblasts. Endocrinology. 1997; 138 1014-1018
44 Rubin C, Turner A S, Mallinckrodt C, Jerome C, McLeod K, Bain S. Mechanical strain, induced noninvasively in the high-frequency domain, is anabolic to cancellous bone, but not cortical bone. Bone. 2002 a; 30 445-452
45 Rubin C, Turner A S, Muller R, Mittra E, McLeod K, Lin W, Qin Y X. Quantity and quality of trabecular bone in the femur are enhanced by a strongly anabolic, noninvasive mechanical intervention. J Bone Miner Res. 2002 b; 17 349-357
46 Ryder K D, Duncan R L. Parathyroid hormone enhances fluid shear-induced [Ca²⁺]_i signaling in osteoblastic cells through activation of mechanosensitive and voltage-sensitive Ca²⁺ channels. J Bone Miner Res. 2001; 16 240-248
47 Sakai K, Mohtai M, Shida J, Hatimaya K, Benvenuti S, Brandi M L, Kukita T, Iwamoto Y. Fluid shear increases interleukin-11 expression in human osteoblast-like cells: its role in osteoclast induction. J Bone Miner Res. 1999; 14 2089-2098
48 Salazar E P, Rozengurt E. Src family kinases are required for integrin-mediated but not for G protein-coupled receptor stimulation of focal adhesion kinase autophosphorylation at Tyr-397. J Biol Chem. 2001; 276 17788-17795
49 Schmidt C, Pommerenke H, Dürr F, Nebe F, Rychly J. Mechanical stressing of integrin receptors induces enhanced tyrosine phosphorylation of cytoskeletally anchored proteins. J Biol Chem. 1998; 273 5081-5085
50 Schneider G B, Zaharias R, Stanford C. Osteoblast integrin adhesion and signaling regulate mineralization. J Dent Res. 2001; 80 1540-1544
51 Smalt R, Mitchell F, Howard R, Chambers T J. Mechanical strain versus wall shear stress as the stimulus to bone cells in mechanical loading. J Bone Miner Res. 1996; 11 S265-(Abstr.)
52 Srinivasan S, Weimer D A, Agans S C, Bain S D, Gross T S. Low-magnitude mechanical loading becomes osteogenic when rest is inserted between each load cycle. J Bone Miner Res. 2002; 17 1613-1620
53 Sun H B, Yokata H. Altered mRNA level of matrix metalloproteinase-13 in MH7 A synovial cells under mechanical loading and unloading. Bone. 2001; 28 399-403
54 Tang L Y, Cullen D M, Yee J A, Jee W SS, Kimmel D B. Prostaglandin E2 increases the skeletal response to mechanical loading. J Bone Miner Res. 1997; 12 276-282
55 Toma C D, Ashkar S, Gray M L, Schaffer J L, Gerstenfeld L C. Signal transduction of mechanical stimuli is dependent on microfilament integrity: identification of osteopontin as a mechanically induced gene in osteoblasts. J Bone Miner Res. 1997; 12 1626-1636
56 Vico L, Chappard D, Alexandre C, Palle S, Minaire P, Riffat G, Morukov B, Rakhmanov S. Effects of a 120 day period of bed-rest on bone mass and bone cell activities in man: attempts at countermeasure. Bone Miner. 1987; 5 383-394
57 Wadhwa S, Godwin S L, Peterson D R, Epstein M A, Raisz L G, Pilbeam C C. Fluid flow induction of cyclo-oxygenase 2 gene expression in osteoblasts is dependent on an extracellular signal-regulated kinase signaling pathway. J Bone Miner Res. 2002; 17 266-274
58 Wary K K, Dans M, Mariotti A, Giancotti F G. Biochemical analysis of integrin-mediated Shc signaling. Methods Mol Biol. 1999; 129 35-49
59 Wennerberg K, Armulik A, Sakai T, Karlsson M, Fässler R, Schaefer E M, Mosher D F, Johansson S. The cytoplasmic tyrosines of integrin subunit beta1 are involved in focal adhesion kinase activation. Mol Cell Biol. 2000; 20 5758-5765
60 Zaman G, Pitsillides A A, Rawlinson S CF, Suswillo R FL, Mosley J R, Cheng M Z, Platts L AM, Hukkanen M, Polak J M, Lanyon L E. Mechanical strain stimulates nitric oxide production by rapid activation of endothelial nitric oxide synthase in osteocytes. J Bone Miner Res. 1999; 14 1123-1131
61 Zaman G, Suswillo R FL, Cheng M Z, Tavares L A, Lanyon L E. Early responses to dynamic strain change and prostaglandins in bone derived cells in culture. J Bone Miner Res. 1997; 12 769-777

Dr. Christian Kasperk

Ruprecht-Karls-University
Department of Medicine I/Endocrinology and Metabolism

Luisenstraße 5

69115 Heidelberg

Germany

Phone: + 496221568604

Fax: + 49 62 21 56 43 59

Email: Christian.Kasperk@med.uni-heidelberg.de