Up-regulation of the Wnt, Estrogen Receptor, Insulin-like Growth Factor-I, and Bone Morphogenetic Protein Pathways in C57BL/6J Osteoblasts as Opposed to C3H/HeJ Osteoblasts in Part Contributes to the Differential Anabolic Response to Fluid Shear

C57BL/6J (B6), but not C3H/HeJ (C3H), mice responded to mechanical loading with an increase in bone formation. A 30-min steady fluid shear of 20 dynes/cm2 increased [3H]thymidine incorporation and alkaline phosphatase activity and up-regulated the expression of early mechanoresponsive genes (integrin β1 (Igtb1) and cyclooxygenase-2 (Cox-2)) in B6 but not C3H osteoblasts, indicating that the differential mechanosensitivity was intrinsic to osteoblasts. In-house microarray analysis with 5,500 gene fragments revealed that the expression of 669 genes in B6 osteoblasts and 474 genes in C3H osteoblasts was altered 4 h after the fluid shear. Several genes associated with the insulin-like growth factor (IGF)-I, the estrogen receptor (ER), the bone morphogenetic protein (BMP)/transforming growth factor-β, and Wnt pathways were differentially up-regulated in B6 osteoblasts. In vitro mechanical loading also led to up-regulation of these genes in the bones of B6 but not C3H mice. Pretreatment of B6 osteoblasts with inhibitors of the Wnt pathway (endostatin), the BMP pathway (Noggin), or the ER pathway (ICI182780) blocked the fluid shear-induced proliferation. Inhibition of integrin and Cox-2 activation by echistatin and indomethacin, respectively, each blocked the fluid shear-induced up-regulation of genes associated with these four pathways. In summary, up-regulation of the IGF-I, ER, BMP, and Wnt pathways is involved in mechanotransduction. These four pathways are downstream to the early mechanoresponsive genes, i.e. Igtb1 and Cox-2. In conclusion, differential up-regulation of these anabolic pathways may in part contribute to the good and poor response, respectively, in the B6 and C3H mice to mechanical loading. C57BL/6J (B6), but not C3H/HeJ (C3H), mice responded to mechanical loading with an increase in bone formation. A 30-min steady fluid shear of 20 dynes/cm2 increased [3H]thymidine incorporation and alkaline phosphatase activity and up-regulated the expression of early mechanoresponsive genes (integrin β1 (Igtb1) and cyclooxygenase-2 (Cox-2)) in B6 but not C3H osteoblasts, indicating that the differential mechanosensitivity was intrinsic to osteoblasts. In-house microarray analysis with 5,500 gene fragments revealed that the expression of 669 genes in B6 osteoblasts and 474 genes in C3H osteoblasts was altered 4 h after the fluid shear. Several genes associated with the insulin-like growth factor (IGF)-I, the estrogen receptor (ER), the bone morphogenetic protein (BMP)/transforming growth factor-β, and Wnt pathways were differentially up-regulated in B6 osteoblasts. In vitro mechanical loading also led to up-regulation of these genes in the bones of B6 but not C3H mice. Pretreatment of B6 osteoblasts with inhibitors of the Wnt pathway (endostatin), the BMP pathway (Noggin), or the ER pathway (ICI182780) blocked the fluid shear-induced proliferation. Inhibition of integrin and Cox-2 activation by echistatin and indomethacin, respectively, each blocked the fluid shear-induced up-regulation of genes associated with these four pathways. In summary, up-regulation of the IGF-I, ER, BMP, and Wnt pathways is involved in mechanotransduction. These four pathways are downstream to the early mechanoresponsive genes, i.e. Igtb1 and Cox-2. In conclusion, differential up-regulation of these anabolic pathways may in part contribute to the good and poor response, respectively, in the B6 and C3H mice to mechanical loading. Mechanical loading is essential for maintenance of skeletal architectural integrity. Loading stimulates bone formation and suppresses bone resorption, leading to an overall increase in bone mass (1Hillam R.A. Skerry T.M. J. Bone Miner. Res. 1995; 10: 683-689Crossref PubMed Scopus (158) Google Scholar), whereas unloading results in an overall decrease in bone mass, because of an inhibition of formation along with an increase in resorption (2Bikle D.D. Halloran B.P. J. Bone Miner. Metab. 1999; 17: 233-244Crossref PubMed Scopus (181) Google Scholar). Loading produces strains in the mineralized matrix of bone, which generates interstitial fluid flow through lacunar/canalicular spaces (3Hillsley M.V. Frangos J.A. Biotechnol. Bioeng. 1994; 43: 573-581Crossref PubMed Scopus (245) Google Scholar). This fluid flow exerts a shear stress at surfaces of osteoblasts and osteocytes lining these spaces, which generates biochemical signals to produce biological effects. Multiple interacting signaling pathways are involved in translating the fluid shear signals into biological effects in bone cells (4Kapur S. Baylink D.J. Lau K.-H.W. Bone (NY). 2003; 32: 241-251Crossref PubMed Scopus (296) Google Scholar), and these pathways are collectively referred to as the mechanotransduction mechanism. Mechanical loading is a key regulatory process for bone mass and strength (5Frost H.M. Metab. Bone Dis. and Relat. Res. 1982; 4: 217-229Abstract Full Text PDF PubMed Scopus (94) Google Scholar). Knowledge of the mechanotransduction mechanism would not only yield information about the mechanical stimulation of bone formation but would also provide insights into the pathophysiology of osteoporosis and other bone-wasting diseases. There is increasing evidence that genetics play a major part in determining the bone response to mechanical loading. Studies from our group (6Kodama Y. Umemura Y. Nagasawa S. Beamer W.G. Donahue L.R. Rosen C.R. Baylink D.J. Farley J.R. Calcif. Tissue Int. 2000; 66: 298-306Crossref PubMed Scopus (139) Google Scholar, 7Kodama Y. Dimai H.P. Wergedal J. Sheng M. Malpe R. Kutilek S. Beamer W. Donahue L.R. Rosen C. Baylink D.J. Farley J. Bone (NY). 1999; 25: 183-190Crossref PubMed Scopus (86) Google Scholar) and others (8Akhter M.P. Cullen D.M. Pedersen E.A. Kimmel D.B. Recker R.R. Calcif. Tissue Int. 1998; 63: 442-449Crossref PubMed Scopus (144) Google Scholar, 9Robling A.G. Turner C.H. Bone (NY). 2002; 31: 562-569Crossref PubMed Scopus (178) Google Scholar) demonstrate that C57BL/6J (B6) 2The abbreviations used are: B6, C57BL/6J inbred mice; ALP, alkaline phosphatase; BMP, bone morphogenetic protein; C3H, C3H/HeJ inbred mice; Cox-2, cyclooxygenase-2; ER, estrogen receptor; Erk1/2, extracellular signal-regulated kinases 1/2; pErk1/2, phosphorylated Erk1/2; EST, expressed sequence tag; IGF-I, insulin-like growth factor-I; TGF-β, transforming growth factor-β; Wnt, wingless- and int-related protein. 2The abbreviations used are: B6, C57BL/6J inbred mice; ALP, alkaline phosphatase; BMP, bone morphogenetic protein; C3H, C3H/HeJ inbred mice; Cox-2, cyclooxygenase-2; ER, estrogen receptor; Erk1/2, extracellular signal-regulated kinases 1/2; pErk1/2, phosphorylated Erk1/2; EST, expressed sequence tag; IGF-I, insulin-like growth factor-I; TGF-β, transforming growth factor-β; Wnt, wingless- and int-related protein. inbred mice responded to in vivo mechanical loading with an increased bone formation, but C3H/HeJ (C3H) mice showed no such response. We postulate that the differential osteogenic response to mechanical stress in B6 and C3H inbred strains of mice is intrinsic to bone cells and that comparative global gene expression profiling studies in osteoblasts derived from this pair of inbred mouse strains in response to fluid shear could provide information concerning potential signaling pathways involved in the mechanical stimulation of bone formation. This would also yield important information about the identity of mechanosensitivity genes that determine the good and poor mechanical response in bone formation, respectively, in B6 and C3H mice. The objectives of this study were 4-fold and are as follows: 1) to confirm that the differential anabolic response to mechanical loading in B6 and C3H strains of mice is intrinsic to osteoblasts, using an in vitro fluid flow shear stress model as a surrogate of mechanical loading (4Kapur S. Baylink D.J. Lau K.-H.W. Bone (NY). 2003; 32: 241-251Crossref PubMed Scopus (296) Google Scholar); 2) to perform in-house microarray analyses in isolated B6 and C3H osteoblasts to identify potential signaling pathways that in part contribute to the differential osteogenic response; 3) to confirm that the pathways-of-interest are essential for fluid shear-induced cell proliferation; and 4) to determine the relationship between the pathways-of-interest and the early mechanoresponsive gene products, such as integrins and cyclooxygenase-2 (Cox-2). Materials—Tissue culture plasticware was obtained from Falcon (Oxnard, CA). Dulbecco's modified Eagle's medium was from Mediatech, Inc. (Herndon, VA). Bovine calf serum was from HyClone (Logan, UT). Trypsin and EDTA were products of Irvine Scientific (Santa Ana, CA). [3H]Thymidine (48 Ci/mmol) was obtained from Research Products International (Mount Prospect, IL). Anti-pErk1/2, anti-pan-Erk1/2, anti-β-catenin, anti-integrin β1, anti-Cox-2, and anti-actin antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA), Upstate Biotechnology, Inc. (Lake Placid, NY), or BD Transduction Laboratories. Echistatin, endostatin, and indomethacin were products of Sigma. ICI182780 was purchased from Tocris (Ellisville, MO), and Noggin was obtained from R&D Systems (Minneapolis, MN). Other chemicals were of molecular biology grade and were from Fisher or Sigma. Cell Culture and Fluid Shear Stress Experiments—Osteoblasts, isolated from calvarias or long bones of 8-week-old B6 and C3H mice by collagenase digestion as described previously for neonatal calvarial osteoblasts (10Sheng M.H.-C. Lau K.-H. W. Beamer W.G. Baylink D.J. Wergedal J.E. Bone (NY). 2004; 35: 711-719Crossref PubMed Scopus (39) Google Scholar), were maintained in Dulbecco's modified Eagle's medium supplemented with 10% bovine calf serum. Pilot studies indicated that cell passage, up to passage 7, had no significant effects on the responsiveness of primary B6 mouse osteoblasts to fluid shear stress with respect to [3H]thymidine incorporation, alkaline phosphatase (ALP) specific activity, and Erk1/2 phosphorylation. Accordingly, cells of passages 3-6 were used in this study. 50,000 cells were plated on each glass slide. At ∼80% confluency, the cells were serum-deprived for 24 h and subjected to a steady fluid shear stress of 20 dynes/cm2 for 30 min in the Cytodyne flow chamber as described previously (4Kapur S. Baylink D.J. Lau K.-H.W. Bone (NY). 2003; 32: 241-251Crossref PubMed Scopus (296) Google Scholar). This dosage of fluid shear stress is believed to be within the physiologically relevant range of laminar shear stress produced by the circulation (11He X. Ku D.N. Moore Jr., J.E. Annu. Biomed. Eng. 1993; 21: 45-49Crossref PubMed Scopus (51) Google Scholar). Replicate glass slides of cells were placed in a parallel flow chamber but without the fluid shear stress as static controls in each experiment. To test the potential involvement of a given signaling pathway-of-interest, cells were pretreated with a specific inhibitor of the pathway-of-interest (i.e. ICI182780 for the estrogen receptor (ER) pathway, endostatin for the canonical wingless- and int-related protein (Wnt) pathway, and Noggin for the bone morphogenetic protein (BMP) pathway), for 24 h prior to the fluid shear stress. To assess the role of integrin activation and Cox-2 on the up-regulation of these pathways, cells were pretreated with echistatin or indomethacin, respectively, for 2 h prior to the fluid shear stress. [3H]Thymidine Incorporation Assay—Cell proliferation was assessed by [3H]thymidine incorporation during the final 6 h of the 24-h post-exposure to fluid shear as described previously (4Kapur S. Baylink D.J. Lau K.-H.W. Bone (NY). 2003; 32: 241-251Crossref PubMed Scopus (296) Google Scholar, 12Lau K.-H.W. Lee M.Y. Linkhart T.A. Mohan S. Vermeiden J. Liu C.C. Baylink D.J. Biochim. Biophys. Acta. 1985; 840: 56-68Crossref PubMed Scopus (34) Google Scholar). Cellular ALP Specific Activity Assay—Osteoblast differentiation was measured by the increase in the specific activity of ALP 24 h post-exposure to the shear stress as described previously (4Kapur S. Baylink D.J. Lau K.-H.W. Bone (NY). 2003; 32: 241-251Crossref PubMed Scopus (296) Google Scholar, 13Farley J.R. Jorch U.M. Arch. Biochem. Biophys. 1983; 221: 477-488Crossref PubMed Scopus (36) Google Scholar). The ALP-specific activity (i.e. normalized against cellular protein content) was reported to adjust for the difference in the cell number because of the increase in cell proliferation in response to fluid shear. Western Immunoblot Assays—Cellular integrin β1, Cox-2, and β-catenin were determined by Western immunoblot assays were performed as described previously (4Kapur S. Baylink D.J. Lau K.-H.W. Bone (NY). 2003; 32: 241-251Crossref PubMed Scopus (296) Google Scholar) using respective commercial polyclonal antibodies and normalized against each corresponding cellular actin level. The relative cellular phosphorylated Erk1/2 (pErk1/2) level (an index of Erk1/2 activation) was determined with the phospho-specific antibody against pErk1/2 and normalized against corresponding total Erk1/2 level, determined with anti-pan-Erk1/2 polyclonal antibody. RNA Purification—Total RNA of cells on each slide was extracted with Qiagen mini RNA kit (Qiagen, Valencia, CA). The purity and integrity of each RNA sample was confirmed with Bio-analyzer (Agilent, Palo Alto, CA). Only undegraded RNA samples were used in this study. In-house Microarray Hybridization and Data Analysis—For the preparation of our in-house microarray chips, cDNA inserts of 5,500 cDNA clones of mouse, rat, human, or monkey genes or ESTs (largely mouse and human genes) were isolated, purified, and evaluated with agarose gel electrophoresis. The microarrays were printed on aminosilane-coated microscope slides (Corning, NY) with a GMS 417 Arrayer (Genetic MicroSystems, Santa Clara, CA). Six replicates of each clone were printed on each slide. DNA was fixed to the slides by baking at 80 °C for 2 h. The experimental strategy and analyses of the microarray experiment are described briefly as follows. Primary osteoblasts isolated from B6 or C3H inbred strain of mice were plated on glass slides and subjected to a 30-min steady shear stress as described above. Replicate plates of B6 or C3H osteoblasts were placed in the flow chamber without the fluid shear as the static control. Four hours after the fluid shear, total RNA was isolated. cDNA synthesized from 1 μg of total RNA of cells received the fluid shear, and corresponding static control cells were each fluorescently labeled with Cy5 and Cy3, respectively, as previously described (14Li X. Mohan S. Gu W. Baylink D.J. Mamm. Genome. 2001; 12: 52-59Crossref PubMed Scopus (68) Google Scholar). The microarray hybridization was performed as described previously (14Li X. Mohan S. Gu W. Baylink D.J. Mamm. Genome. 2001; 12: 52-59Crossref PubMed Scopus (68) Google Scholar). The slide was scanned using a ScanArray 4000 scanner (GSI Lumonics, San Jose, CA). The fluorescent images were acquired using ScanArray software (version 2.1; GSI Lumonics), and data were analyzed using GeneSpring Image Analysis program (Silicon Genetics, San Jose, CA). Each array spot was individually inspected using the Gene-Spring Image Analysis program. The microarray analysis was repeated in osteoblasts of four pairs of B6/C3H mice. Statistically significant differences in gene expression between each pair of stressed and corresponding static control samples was analyzed using Lowess Normalization and paired t test. Differences of p < 0.05 were considered significant. Only known mouse genes were analyzed further. Because the gene annotation or accession numbers of many of the known mouse genes on our array were missing, the computer-based gene ontology and pathway analyses were not performed. Tentative classification of gene functions was determined manually based on information available on the PubMed data base. Real Time PCR Analyses—Real time PCR was carried out with the SYBR Green method on the MJ Research DNA Engine Opticon® 2 System (Waltham, MA). The purified total RNA was used to synthesize cDNA by reverse transcription using random hexamer primers and Superscript II reverse transcriptase (Invitrogen). The cDNA was then subjected to real time PCR amplification using the gene-specific primers listed in Table 1. The primers were designed with the IDT Vector NTI software (Coralville, IA). An aliquot (25 μl) of reaction volume (consisted of 2× (12.5 μl) QuantiTect SYBR Green PCR master mix, which contained the Hot Start Taq polymerase (Qiagen), 0.5 μm of primers, and 1-5 μl of cDNA template) was used in each assay. The PCR conditions consisted of an initial 10-min hot start at 95 °C, followed by 40 cycles of denaturation at 95 °C for 30 s, annealing, and extension at appropriate temperature (50-72 °C) (see Table 1) for 30 s, and a final step of melting curve analysis from 60 to 95 °C. Each reaction was performed in triplicate. The data were analyzed using Opticon® Monitor Software 2.0. Data normalization was performed against β-actin, and the normalized values were used to calculate the relative fold change between the control and the experiment groups by the threshold cycle method.TABLE 1List of primer sets used in the real-time PCR amplification reactionsGenePrimer sequencesAnnealing temperatureExtension temperature°Cβ-ActinForward primer, 5′-CAG GCA TTG CTG ACA GGA TG-3′5672Reverse primer, 5′-TGC TGA TCC ACA TCT GCT GG-3′Tgfb1Forward primer, 5′-CGG CAG CTG TAC ATT GAC TT-3′5372Reverse primer, 5′-TGT GTT GGT TGT AGA GGG CA-3′Ctnnb1Forward primer, 5′-GAC TCA CGC AGT GAA GAA TG-3′5072Reverse primer, 5′-GCT GTA GCA GGT TCA CTA GA-3′Bmpr1Forward primer, 5′-GCT TAT TCT GCT GCT TGT GGG-3′5372Reverse primer, 5′-ATT TAA CAG CTA GGC CCA GG-3′Igf1rForward primer, 5′-GCC AAC AAG TTC GTC CAC AG-3′5672Reverse primer, 5′-CCG AAG GAC CAG ACA TCA GA-3′Wnt1Forward primer, 5′-GGT GTT GCG GTT CCT GAT GT-3′5472Reverse primer, 5′-TCC GAG GCA GAG ACA AGG AG-3′Wnt3aForward primer, 5′-ATA GCC TGC ATC CGC TCT GA-3′5472Reverse primer, 5′-TGG TGA CCA TTG CCT CAA CA-3′Wnt5aForward primer, 5′-AAC TGC AGC ACA GTG GAC AA-3′5472Reverse primer, 5′-TAG TCG ATG TTG TCT CCG CA-3′AxinForward primer, 5′-TCT GGA TAC CTG CCC ACT TT-3′5472Reverse primer, 5′-TGC CTT CGT TGT ACC GTC TA-3′Lef1Forward primer, 5′-ACG GAC AGT GAC CTA ATG CA-3′5472Reverse primer, 5′-TCT CCT TTA GCG TGC ACT CA-3′Lrp5Forward primer, 5′-ACA CTA TAT CCG CCG ATC CT-3′5372Reverse primer, 5′-GAC TGG TGC TGT AGT CAC TG-3′Esr1Forward primer, 5′-ATG TGC AGG AGG CAG ACA TT-3′5672Reverse primer, 5′-TGG AGC CTG CTT GGA GTT AT-3′Ncoa1Forward primer, 5′-GCA CAG CCA GGA GTG TAC AA-3′5672Reverse primer, 5′-GAC GAG AGC TGG TTG CAG TA-3′Dlx1Forward primer, 5′-GGA CCG GAC CAG ACT CTC AT-3′5672Reverse primer, 5′-GTG GCT CAG ACC TGG TGA CT-3′c-FosForward primer, 5′-CCT GAG GTC TTT CGA CAT GTG GAA-3′5672Reverse primer, 5′-AAG AGA GCA AGA AGG TGG TCG CAT-3′ Open table in a new tab In Vivo Mechanical Loading Model—We adapted the four-point bending exercise regimen, originally developed by Akhter et al. (15Akhter M.P. Raab D.M. Turner C.H. Kimmel D.B. Recker R.R. J. Biomech. 1992; 25: 1241-1246Crossref PubMed Scopus (83) Google Scholar) on rat tibia, as the in vivo mechanical loading model for mouse tibia as described previously (16Kesavan C. Mohan S. Oberholtzer S. Wergedal J.E. Baylink D.J. J. Appl. Physiol. 2005; 99: 1951-1957Crossref PubMed Scopus (52) Google Scholar). Briefly, the four-point bending device (Instron, Canton, MA) consisted of two upper vertically movable points covered with rubber pads (4-mm apart), and two 12-mm lower non-movable points covered with rubber pads. During the bending exercise, the two upper pads touched the lateral surface of the tibia through overlying muscle and soft tissue, whereas the lower pads touched the medial surface of the proximal and distal parts of the tibia. The loading protocol consisted of a 9-newton load at a frequency of 2 Hz for 36 cycles, and the exercise was performed once daily. The right tibia was subjected to the loading exercise, and the left tibia was used as an internal unloaded control. Upon anesthesia with halothane, the ankle of the tibia was positioned on the second lower immobile points of the Instron equipment, such that the region of the tibia loaded did not vary from mouse to mouse. The loading was applied for 6 days/week with 1 day of rest for 2 weeks. Forty eight h after the final loading, mice were sacrificed. The marrow-flushed tibias were stored at -80 °C until RNA extraction. The animal protocol was approved by the Institutional Animal Care and Use Committee of the J. L. Pettis Memorial Veterans Affairs Medical Center. One of the potential limitations of this model is that force applied over the soft tissues may have local bruising effects that could result in inflammation. Accordingly, extra efforts were taken to minimize the bruising effect of loading on soft and hard tissues by changing the rubber pads frequently. We also looked for histological evidence for inflammation (i.e. presence of inflammatory cells and/or blood clots) in the loaded muscles and bones after the loading regimen in several mice in preliminary experiments, and we found no evidence for the presence of lymphocytes or other inflammatory cells in the muscle and bone tissues at the loading sites in all samples examined. We also found no evidence of blood clotting at the loading limb. RNA Extraction from Bones—Briefly, bones were pulverized in liquid nitrogen. Total RNA was purified with Trizol reagent (Invitrogen), followed by RNeasy columns (Qiagen). The quality and quantity of RNA were analyzed using Bio-analyzer and Nano-drop instrumentation, respectively. Only good quality RNAs were used for subsequent real time PCR analyses. Bone Histomorphometry—Both the loaded and unloaded (control) tibiae of B6 and C3H mice were removed, after euthanasia, and fixed with 10% cold neutral buffered formalin on ice. The fixed bones were then rinsed free of formalin, defleshed, and embedded in methyl methacrylate (17Sheng M.H.-C. Baylink D.J. Beamer W.G. Donahue L.R. Lau K.-H. W. Wergedal J.E. Bone (NY). 2002; 30: 486-491Crossref PubMed Scopus (35) Google Scholar). Thick (0.5 mm) cross-sections were cut from the middiaphysis of the tibia with a wire saw (Delaware Diamond Knives), lightly ground, and stained with Goldner's trichrome stain for mineralized bone. The stained bone slices were mounted in Fluoromount-G (Fisher) and examined under an Olympus BH-2 fluorescence/bright field microscope. Statistical Analysis—Results are shown as mean ± S.D. with 3-6 replicates or repeat measurements. Statistical significance was determined with two-tailed Student's t test, and the difference was significant at p < 0.05. Effects of Fluid Shear Stress on the Proliferation, Differentiation, and Expression of Early Mechanoresponsive Genes in Primary Osteoblasts of B6 and C3H Inbred Strains of Mice—The 30-min steady fluid shear stress of 20 dynes/cm2 significantly increased (∼2-fold each) the [3H]thymidine incorporation (Fig. 1A) (an index of cell proliferation) and ALP-specific activity (Fig. 1B) (a marker of osteoblast differentiation) of B6 osteoblasts. No such response was seen in C3H osteoblasts. Mechanical stimulation, including fluid shear stress, has been shown to up-regulate several early mechanoresponsive genes, such as integrins and Cox-2, within minutes in bone cells (18Pavalko F.M. Chen N.X. Turner C.H. Burr D.B. Atkinson S. Hsieh Y.F. Qiu J. Duncan R.L. Am. J. Physiol. 1998; 275: C1591-C1601Crossref PubMed Google Scholar). Thus, we evaluated whether there was also a differential response to fluid shear in the expression of integrin β1 (Igtb1) and Cox-2 in C3H and B6 osteoblasts. Fig. 1C shows that while the 30-min steady fluid shear stress significantly increased the cellular integrin β1 protein level in B6 osteoblasts by ∼2-fold 10 min after the stress, the same stress had no effect in C3H osteoblasts. Similarly, the fluid shear significantly increased Cox-2 protein expression by >2-fold in B6 osteoblasts but not in C3H osteoblasts (Fig. 1D). Fig. 1, C and D, also shows that the basal cellular integrin β1 and Cox-2 protein levels in C3H osteoblasts were severalfold higher than those in B6 osteoblasts. The significance of the higher basal expression of these two early mechanoresponsive genes in C3H osteoblasts is unclear. Nevertheless, these results clearly indicate that the osteogenic response to mechanical loading in this pair of inbred strains of mice is intrinsic to osteoblasts. In-house Microarray Analysis of Shear Stress-mediated Changes in Gene Expression in Primary Osteoblasts of B6 and C3H Mice—Microarray analysis was performed with RNAs isolated from primary B6 or C3H osteoblasts 4 h after the 30-min fluid shear, using the in-house chips, which contained 5,500 genes or ESTs of mouse, human, and several other species. As schematically summarized in Fig. 2, of the 5,500 genes or ESTs on the microarray chip, the expression of 669 genes or ESTs in B6 osteoblasts (360 up-regulated and 309 down-regulated) and that of 474 genes or ESTs in C3H osteoblasts (212 up-regulated and 262 down-regulated) was significantly (p < 0.05) affected by the shear stress. Among the affected gene fragments, 286 were up-regulated and 228 down-regulated in B6 osteoblasts only, and 138 of them were up-regulated and 181 were down-regulated in C3H osteoblasts only. Seventy three genes or ESTs were up-regulated and 80 were down-regulated in both B6 and C3H osteoblasts. The fibronectin (Fn1) gene was up-regulated in B6 osteoblasts but down-regulated in C3H osteoblasts. Conversely, the solute carrier family 34 (sodium phosphate), member 2 (Slc34a2) gene was down-regulated in B6 osteoblasts but up-regulated in C3H osteoblasts. Although the relative changes in the expression level of these genes were mostly <3-fold, all of the changes were statistically significant (p < 0.05). Because we sought to identify potential signaling pathways that might contribute to the differential osteogenic response in this pair of mouse strains, subsequent analyses were focused on the known mouse genes whose expression was affected differentially in B6 osteoblasts, with an emphasis on the up-regulating genes. Accordingly, 53 known mouse genes were up-regulated and 20 known genes were down-regulated in both B6 and C3H osteoblasts (supplemental Table 1). The up-regulated genes included a number of key regulator genes of cell proliferation and differentiation in both B6 and C3H osteoblasts, including several growth factor genes (i.e. Tgfb1, Vegfd, Igf2, Pdgfa, Fgf1, and Op2/Bmp8b), receptor genes (Thr, Bmpr1a, Pthr, Esr2, Rarg, Fnrb, Osmr, Ifngr, and Tnfr), vitamin D metabolism genes (i.e. Cyp27b1), small G-protein genes (i.e. Ran and Era1), and several inhibitory transcription factor genes of osteoblast differentiation (i.e. M-twist, Id-2, and Dermo-1). Because these genes were up-regulated in both C3H and B6 osteoblasts, these mechanoresponsive genes were likely to be upstream to the mechanosensitivity genes responsible for the differential anabolic response to fluid shear between B6 and C3H osteoblasts. The expression of 88 known mouse genes (50 up-regulated and 33 down-regulated) was altered only in C3H osteoblasts (supplemental Table 2). Some of the up-regulated genes were growth factor and receptor genes (Csf1, Tgfbr1, Igfbp2, and Fgfr), transcription factor genes (Hox8.1/Msx2, and c-Myc), signal transduction genes (Pld, Hic5, Itgb4bp, and Emk2), and intracellular transport and trafficking genes (Gs15, Cacnb3, Snx3, and Atp6d). The shear stress also up-regulated Pges and Bcl genes in C3H osteoblasts but not in B6 osteoblasts. Because C3H osteoblasts did not respond anabolically to fluid shear, these genes were not analyzed further. The expression of 129 known mouse genes was up-regulated in B6 osteoblasts only (Table 3). Consistent with an anabolic response to the fluid shear in B6 osteoblasts and not C3H osteoblasts, the fluid shear differentially up-regulated in B6 osteoblasts a number of genes associated with osteoblast proliferation and differentiation. These genes include, but are not limited to, bone growth factor, receptor, and associated genes (i.e. Tgfb2, Bmp4, Fgf6, Kgf/Fgf7, Pdgfc, Igf1r, Ghr, Bmpr2, Igfbp5, and Wnt5a), cytokines, and receptor genes (i.e. Osm, Il4, Il6, Il8, and Il6r), Esr1, genes involved in protein and RNA synthesis, DNA synthesis, as well as cell proliferation. A number of energy and cell metabolism genes, intracellular transport and trafficking genes, as well as oxidative stress-responsive genes, such as Hsc70, Osp94, and p47Phox, were also up-regulated in B6 but not C3H osteoblasts. Similarly, a large number of transcription factors and signal transduction molecules were differentially up-regulated in B6 osteoblasts.TABLE 3Real time PCR analyses of in vivo gene expression of the Wnt, BMP/ TGF-β, IGF-I, and ER signaling pathways in response to the four-point bending exercise regimen in tibia of B6 inbred strain of mice as opposed to that of C3H inbred strain of mice (mean ± S.D., n = 6 for each mouse strain)GeneB6 mice, fold changesC3H mice, fold changesThe canonical Wnt signaling pathway genesWnt11.81 ± 0.68ap < 0.05.0.74 ± 0.45Wnt3a4.25 ± 2.12bp < 0.01.3.10 ± 2.26Lrp59.08 ± 2.76bp < 0.01.3.22 ± 1.59Ctnnb15.51 ± 2.62bp < 0.01.1.29 ± 0.37The BMP/TGF-β signaling pathway genesTgfb12.10 ± 0.80bp < 0.01.0.53 ± 0.34Bmpr13.23 ± 0.96bp < 0.01.1.64 ± 0.78Dlx13.06 ± 1.21ap < 0.05.Not detectableThe IGF-I signaling pathway genesIgfr12.74 ± 1.22ap < 0.05.1.51 ± 1.08c-Fos3.73 ± 1.65bp < 0.01.0.98 ± 0.65The ER signaling pathway genesEsr13.17 ± 1.27bp < 0.01.1.55 ± 0.53Ncoa12.05 ± 0.61bp < 0.01.1.56 ± 0.85a p < 0.05.b p < 0.01. Open table in a new tab Up-regulation of the Expression of Genes of Four Anabolic Signa