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/cm(2) increased [(3)H]thymidine incorporation and alkaline phosphatase activity and up-regulated the expression of early mechanoresponsive genes (integrin beta1 (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-beta, 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.     

Genetics and bone. Using the mouse to understand man

The rationale for use of inbred strains of mice in bone research is well recognized and includes: a) practical factors (economics of scale, rapid development of adult status, pre-existing knowledge, down-sized technologies) and b) proven methodologies for genetic studies (polygenic trait analyses, mapping tools, genomic sequencing, methods for gene manipulation). Initial investigations of inbred strains of mice showed that femoral and lumbar vertebral volumetric bone mineral density (BMD, mg/mm(3)) by pQCT varied in excess of 50% for femurs and 9% in vertebral BMD. Two strains - low BMD C57BL/6J (B6) mice and high BMD C3H/HeJ (C3H) - were investigated for insights to their BMD diversity. B6C3F2 females derived from intercrossing B6C3F1s were raised to adult skeletal status at 4 months, then necropsied for phenotyping of bone and genotyping of genomic DNA. 1000 F2 females were genotyped for PCR product polymorphisms on all 19 autosomes at approximately 15 cM. Genome wide analyses for genotype-phenotype correlations showed 10 chromosomes (Chrs) carried genes for femoral and 7 Chrs for vertebral BMD. LOD scores ranged from 2.90 to 24.4, and percent of F2 variance accounted for ranged from 1 to 10%. Analyses of main effects revealed both dominant-recessive and additive inheritance patterns. Both progenitor strains carried alleles with positive and negative effects on BMD of each bone sites. A remarkable array of additonal skeletal phenotypes (femur and vertebral geometry, strength measures, serum markers) also proved polygenic in nature, with complex segregation patterns. Verification of BMD quantitative trait loci (QTLs) was undertaken by creating congenic B6 strains carrying individual QTL regions from C3H. Following 6 cycles of backcrossing a QTL-containing region from C3H to the B6 strain, N6F2 congenic strain mice were aged to 4 months, then genotyped for the QTL region and phenotyped for skeletal traits. Comparison of mice homozygous for C3H alleles versus homozygous for B6 alleles in the QTL regions showed that femoral BMD increased or decreased significantly in congenic strains, as was predicted from F2 data. Gender differences specific to BMD QTLs have been revealed, as have more than 30 additional phenotypes associated with cortical and trabecular structural parameters and biomechanical properties.     

A potent modifier of liver cancer risk on distal mouse chromosome 1: linkage analysis and characterization of congenic lines

The C3H/HeJ (C3H) and CBA/J (CBA) mouse strains are classical mouse models of cancer susceptibility, exhibiting high risks for both spontaneous and chemically induced liver cancer. By analysis of backcrosses and intercrosses between C3H or CBA and resistant B6 mice, we have mapped a potent modifier of hepatocellular carcinoma development to distal chromosome 1, linked to the marker D1Mit33 with combined LOD(W) scores of approximately 5.9 (C3H) and 6.5 (CBA). We previously identified this region as one of two that modify susceptibility in the more distantly related C57BR/cdJ (BR) strain. Congenic B6.C3H(D1Mit5-D1Mit17) and B6.BR(D1Mit5-D1Mit17) mice developed significantly more liver tumors than B6 mice did (6- to 13-fold, P < 10(-11), in males; 3- to 4-fold, P < 10(-3), in females). Thus, distal chromosome 1 carries one or more genes that are sufficient to confer susceptibility to liver cancer.     

Genetic analysis of expulsion of adult Trichinella spiralis in NFS, C3H/He, and B10.BR mice

The genetics of T. spiralis rejection from the intestine was examined in inbred mice belonging to three phenotypic categories of expulsion: strong (NFS), intermediate (C3H), and weak (B10.BR). Experiments used various worm doses to analyze the day of worm rejection, defined as the day at which 98% expulsion of the infectious dose occurred. The F1 of NFS (strong) x B10.BR (weak) was a strong responder and the F1 of the cross C3H (intermediate) x B10.BR (weak) was intermediate. Analysis of time of rejection among offspring of the (NFS/B10.BR) x B10.BR backcross showed three segregating phenotypic categories which occurred in a ratio of 1:2:1 strong:intermediate:weak. Segregation analysis of C3H/B10.BR intercross (F2) mice produced a ratio of 3:1, intermediate:weak. The backcross C3H/B10.BR to the C3H parent produced 100% intermediate offspring and the backcross to the B10.BR parent segregated in a 1:1 ratio of intermediate:weak. Taken together the results of both sets of crosses demonstrated that strong responsiveness was a consequence of the additive effects of two dominant genes; either gene by itself conferred intermediate responsiveness. The additive nature of these dominant genes suggested that two distinct processes each lead to the expression of worm expulsion that is phenotypically intermediate and kinetically identical.     

IL-2 production, IL-2 receptor expression, and IL-2 responsiveness of spleen and mesenteric lymph node cells from inbred mice infected with Trichinella spiralis

The in vitro production of IL-2 and IL-2R expression by lymphoid cells of inbred mice of strong (NFS), intermediate (C3H), or weak (B10.BR) in phenotype of Trichinella spiralis (TS) rejection was measured during a primary infection. Maximum production of IL-2 by spleen and mesenteric lymph node (MLN) cells occurred at 5 days postinfection. Cell depletion experiments demonstrated that Lyt-1.2+ T cells were predominantly responsible for in vitro IL-2 production. Cells from strong-responder NFS mice produced more IL-2 than cells from intermediate-responder C3H or weak-responder B10.BR mice. Similarly, after TS infection, NFS mice had significantly more IL-2R expressing MLN cells than B10.BR or C3H MLN cells. All mouse strains displayed a dose-dependent increase in in vitro IL-2 production after infection with 100 to 800 TS. This effect was most pronounced in NFS mice. Limiting dilution analysis of day 5 infected MLN cells demonstrated that the frequency of TS-reactive CD4+ cells was threefold higher in NFS mice than B10.BR and fourfold higher than in C3H mice. Finally, MLN cells taken from infected NFS mice responded to an exogenous source of IL-2, whereas MLN cells from infected C3H or B10.BR mice were unable to do so. We conclude that strong responsiveness in parasite rejection may be related to the amount of IL-2 produced as well as to the capacity of the lymphocytes of each mouse strain to respond to IL-2. Although these differences help explain the strong rejection phenotype of NFS mice, they fail to separate C3H and B10.BR mice where TS-responsive CD4+ precursors, IL-2 production, and dose responsiveness are all lower for the intermediate phenotype (worm rejection) C3H than the weak phenotype B10.BR mice.     

Mechanical loading of mouse caudal vertebrae increases trabecular and cortical bone mass-dependence on dose and genotype

Most in vivo studies addressing the skeletal responses of mice to mechanical loading have targeted cortical bone. To investigate trabecular bone responses also we have developed a caudal vertebral axial compression device (CVAD) that transmits mechanical loads to compress the fifth caudal vertebra via stainless steel pins inserted into the forth and sixth caudal vertebral bodies. Here, we used the CVAD in C57BL/6 (B6) and C3H/Hej (C3H) female mice (15 weeks of age) to investigate whether the effect of regular bouts of mechanical stimulation on bone modeling and bone mass was dependent on dose and genotype. A combined micro-computed tomographic and dynamic histomorphometric analysis was carried out at the end of a 4-week loading regimen (3,000 cycles, 10 Hz, 3 x week) for load amplitudes of 0N, 2N, 4N and 8N. Significant increases in trabecular bone mass of 9 and 21% for loads of 4N and 8N, respectively, were observed in B6 mice. A significant increase of 10% in trabecular bone mass occurred for a load of 8N in the C3H strain. For other loads, no significant increases were detected. Both mouse strains exhibited substantial increases in trabecular bone formation rates for all loads, B6: 111% (2N), 86% (4N), 164% (8N), C3H: 41% (2N), 38% (4N), 141% (8N). Significant decreases in osteoclast number of 146 and 93% for a load of 8N were detected in B6 and C3H mice, respectively. These findings demonstrate that the effect of loading on the structural and functional parameters of bone is dose and genotype dependent. The caudal vertebral loading model established here is proposed for further studies addressing the molecular processes involved in the skeletal responses to mechanical stimuli.     

Leptin Receptor (Lepr) Is a Negative Modulator of Bone Mechanosensitivity and Genetic Variations in Lepr May Contribute to the Differential Osteogenic Response to Mechanical Stimulation in the C57BL/6J and C3H/HeJ Pair of Mouse Strains

This study investigated the role of leptin receptor (Lepr) signaling in determining the bone mechanosensitivity and also evaluated whether differences in the Lepr signaling may contribute to the differential osteogenic response of the C57BL/6J (B6) and C3H/HeJ (C3H) pair of mouse strains to mechanical stimuli. This study shows that a loading strain of ∼2,500 μϵ, which was insufficient to produce a bone formation response in B6 mice, significantly increased bone formation parameters in leptin-deficient ob⁻/ob⁻ mice and that a loading strain of ∼3,000 μϵ also yielded greater osteogenic responses in Lepr-deficient db⁻/db⁻ mice than in wild-type littermates. In vitro, a 30-min steady shear stress increased [³H]thymidine incorporation and Erk1/2 phosphorylation in ob⁻/ob⁻ osteoblasts and db⁻/db⁻ osteoblasts much greater than those in corresponding wild-type osteoblasts. The siRNA-mediated suppression of Lepr expression in B6 osteoblasts enhanced (but in osteoblasts of C3H (the mouse strain with poor bone mechanosensitivity) restored) their anabolic responses to shear stress. The Lepr signaling (leptin-induced Jak2/Stat3 phosphorylation) in C3H osteoblasts was higher than that in B6 osteoblasts. One of the three single nucleotide polymorphisms in the C3H Lepr coding region yielded an I359V substitution near the leptin binding region, suggesting that genetic variation of Lepr may contribute to a dysfunctional Lepr signaling in C3H osteoblasts. In conclusion, Lepr signaling is a negative modulator of bone mechanosensitivity. Genetic variations in Lepr, which result in a dysfunctional Lepr signaling in C3H mice, may contribute to the poor osteogenic response to loading in C3H mice.     

Bone morphogenetic protein receptor type Ia localization causes increased BMP2 signaling in mice exhibiting increased peak bone mass phenotype

Bone morphogenetic protein 2 (BMP2) is a growth factor that initiates osteoblast differentiation. Recent studies show that BMP2 signaling regulates bone mineral density (BMD). BMP2 interacts with BMP receptor type Ia (BMPRIa) and type II receptor leading to the activation of the Smad signaling pathway. BMPRIa must shuttle between distinct plasma membrane domains, enriched of Caveolin‐1 alpha and Caveolin‐1 beta isoforms, and receptor activation occurs in these domains. Yet it remains unknown whether the molecular mechanism that regulates BMP2 signaling is driving mineralization and BMD. Therefore, the B6.C3H‐1‐12 congenic mouse model with increased BMD and osteoblast mineralization was utilized in this study. Using the family image correlation spectroscopy, we determined if BMP2 led to a significant re‐localization of BMPRIa to caveolae of the alpha/beta isoforms in bone marrow stromal cells (BMSCs) isolated from B6.C3H‐1‐12 mice compared to the C57BL/6J mice, which served as controls. The control, C57BL/6J mice, was selected due to only 4 Mb of chromosome 1 from the C3H/HeJ mouse was backcrossed to a C57BL/6J background. Using reporter gene assays, the B6.C3H‐1‐12 BMSCs responded to BMP2 with increased Smad activation. Furthermore, disrupting caveolae reduced the BMP2‐induced Smad signaling in BMSCs isolated from B6.C3H‐1‐12 and C57BL/6J. This study suggests for the first time a regulatory mechanism of BMPRIa signaling at the plasma membrane of BMSCs that (i) associated with genetic differences in the distal Chromosome 1 segment carried by the B6.C3H‐1‐12 congenic and (ii) contributes to increase BMD of the B6.C3H‐1‐12 compared to the C57BL/6J control mice. J. Cell. Physiol. 227: 2870–2879, 2012. © 2011 Wiley Periodicals, Inc.     

Gene expression in lung and basal forebrain during influenza infection in mice

Inbred mice develop strain-dependent changes in sleep during the first few days after inoculation with influenza virus. To identify genes with the potential to differentially modulate sleep under this condition, we performed complementary DNA microarray analysis of both lung and basal forebrain (BF) of infected (I) and uninfected BALB/cByJ (C) and C57BL/6J (B6) mice. This analysis showed significant variation in the expression pattern of 667 and 1217 of the surveyed genes in BF and lung, respectively ( P  < 0.01). Applying the additional criterion of an effect size ≥2, 495 genes differed in expression in lung compared with 204 in BF. In BF, more genes were differentially expressed as a function of mouse strain, whereas in lung, more genes were differentially expressed as a function of health status. Significant alterations in expression after infection were more numerous and robust in BALB/cByJ vs. C57BL/6J mice. Some genes showed significant variation in both tissues as a function of strain or condition, but the changes in general were not parallel. Genes that showed significant and robust variation as a function of strain, health status or tissue included those related to immune function, metabolism, signal transduction, cell cycle regulation, apoptosis and other miscellaneous categories. Different patterns of gene expression in BF of uninfected mice suggest the possibility of fundamental mechanistic differences in pathways that modulate vigilance in these strains, whereas differences in expression of lung of infected mice suggests different peripherally generated sleep-modulatory stimuli in the two strains.     

Genetic differences in BCG-induced resistance to Schistosoma mansoni are not controlled by genes within the major histocompatibility complex of the mouse.

Induction of nonspecific resistance to Schistosoma mansoni infection after the i.v. injection of viable BCG was investigated in outbred mice and a panel of inbred and H-2 congenic strains. Significant protection was induced in CF1, A/J, C57BL/6, C57BL/10, DBA/2, C57BR, and SJL mice. BALB/c mice were not protected whereas CBA and C3H mice expressed intermediate degrees of protection. Expression of the protective phenomenon is not controlled by genes within the MHC as shown by the marked differences in response between BALB/c and DBA/2 (H-2d) as well as between C57BR and C3H (H-2k) mice. H-2 congenic strains with C57BL/10 background (B10.A and B10.D2) were high responders. BALB.B10 mice carrying the high responder (B10) MHC on the nonresponder (BALB/c) background were not protected. The degree of splenic hypertrophy did not correlate with the expression of nonspecific resistance. These results demonstrate that, in addition to controlling specific immune responses, genetic differences influence the nonspecific protective phenomena related to BCG administration as well.     

Differential effect of Bacillus firmus on immune response and enterocyte brush-border enzyme levels in BALB/c and B10.BR mice

A nonpathogenic bacterium of external environment possessing remarkable immunomodulatory activity, Bacillus firmus (BF) inactivated with formaldehyde, was given intragastrically to two genetically different mouse strains BALB/c (H-2d) and B10.BR/SnPh (B10.BR, H-2k) reared in conventional (CV) and B10.BR strain also in germ-free (GF) conditions. Repeated intragastric administration of BF (500 micrograms every other day over two weeks, starting at the age of 3 months) significantly enhanced intestinal IgA levels in CV BALB/c mice but did not affect intestinal IgA in CV B10.BR mice. In GF B10.BR mice, IgG levels in sera and intestinal washings increased after BF administration compared to CV B10.BR mice. In CV BALB/c mice, specific activity of enterocyte brush-border enzymes (lactase, gamma-glutamyltransferase, alkaline phosphatase) decreased after BF treatment; sucrase (sucrose alpha-glucosidase) activity was not affected. On the other hand, in B10.BR mice, specific activity of gamma-glutamyltransferase and dipeptidyl peptidase IV were higher after administration of BF in both CV and GF groups relative to untreated controls. The activities of lactase and glucoamylase (glucan 1,4-alpha-glucosidase) were significantly stimulated only in the group of GF B10.BR mice treated with formolized BF. The stimulation of immunoglobulin production after BF treatment was accompanied by changes in the levels of enterocyte brush-border enzymes; this responsiveness to BF treatment was genetically regulated.     

Day/night food consumption in mice is strain and age-dependent

Food consumption was measured during the day (lights on) and the night (lights off) and compared between one outbred and 9 inbred strains of mice (CBA/Kw, C3H, DBA2, KP, BALB/c, C57BL, B10.Amst, B10.BR, B10.BR Y-del) in age groups 30-60, 60-90, 90-120, and more than 120 days. Outbred mice and animals from B10 sublines ate significantly more during nocturnal darkness. Day and night food consumption was similar in KP animals. In mice from the remaining strains there was an apparent age-related shift from nocturnal towards diurnal eating habits.     

Modulation of F1 cytotoxic potentials by graft-vs-host reaction. Cooperative non-H-2- and H-2D region-gene control of F1 natural resistance to graft-vs-host reaction-associated immunosuppression

Our previous study revealed that in F1 mice raised by crossing C3H/He or AKR/J mice with various H-2-congenic B10-series strains, parental H-2k spleen cells (SC) could not induce the graft-vs-host reaction (GvHR)-associated immunosuppression (GAIS). We also elucidated that a limited number of non-H-2 genes of parental C3H/He or AKR/J mice that had been incorporated into the F1 hybrids determined the F1 resistance to the GAIS, and the present study was done to explore the mechanism implicated in this type of F1 resistance to GAIS. SC from B10.AL mice carrying an rH-2 (K:k I:k S:k D:d) haplotype but not SC from H-2K B10.BR (k k k k) mice induced GAIS of in vitro CTL responses to third-party alloantigens in H-2k/d (C3H/He x B10.D2)F1 recipients mice. Further, SC from H-2k/a (C3H/He x B10.A)F1 mice carrying heterozygous C3H/B10 non-H-2 background but not SC from the same H-2k/a (B10.BR x B10.A)F1 mice but carrying homozygous B10/B10 background induced GAIS in H-2k/d (C3H/He x B10.D2)F1 recipients. Although C3H/He-, B10.BR-, and C3H.OH (d d d k)-SC were incapable of inducing GAIS in (C3H/He x B10.D2)F1 (k/d k/d k/d k/d) recipients, they were all good inducers of GAIS in (C3H.OH x B10.BR)F1 (d/k d/k d/k k/k) recipients. Exactly the same pattern of co-operative non-H-2 AKR and H-2D region-gene control of GAIS was observed on GvHR induced in H-2k/d (AKR/J x B10.D2)F1 recipients. These results suggest that the non-H-2 genes of C3H/He or AKR/J strain inhibit the functional expression of certain antigenic determinant(s) when it is encoded by heterozygous but not homozygous gene(s) linked tightly to H-2D region of k haplotype. Thus, the F1 resistance to GAIS is mediated by immune response of F1 recipients who miss the antigenic determinant(s) against that expressed on cell surface of GvHR-inducing T lymphocytes.     

Tibial loading increases osteogenic gene expression and cortical bone volume in mature and middle-aged mice

There are conflicting data on whether age reduces the response of the skeleton to mechanical stimuli. We examined this question in female BALB/c mice of different ages, ranging from young to middle-aged (2, 4, 7, 12 months). We first assessed markers of bone turnover in control (non-loaded) mice. Serum osteocalcin and CTX declined significantly from 2 to 4 months (p<0.001). There were similar age-related declines in tibial mRNA expression of osteoblast- and osteoclast-related genes, most notably in late osteoblast/matrix genes. For example, Col1a1 expression declined 90% from 2 to 7 months (p<0.001). We then assessed tibial responses to mechanical loading using age-specific forces to produce similar peak strains (-1300 με endocortical; -2350 με periosteal). Axial tibial compression was applied to the right leg for 60 cycles/day on alternate days for 1 or 6 weeks. qPCR after 1 week revealed no effect of loading in young (2-month) mice, but significant increases in osteoblast/matrix genes in older mice. For example, in 12-month old mice Col1a1 was increased 6-fold in loaded tibias vs. controls (p = 0.001). In vivo microCT after 6 weeks revealed that loaded tibias in each age group had greater cortical bone volume (BV) than contralateral control tibias (p<0.05), due to relative periosteal expansion. The loading-induced increase in cortical BV was greatest in 4-month old mice (+13%; p<0.05 vs. other ages). In summary, non-loaded female BALB/c mice exhibit an age-related decline in measures related to bone formation. Yet when subjected to tibial compression, mice from 2-12 months have an increase in cortical bone volume. Older mice respond with an upregulation of osteoblast/matrix genes, which increase to levels comparable to young mice. We conclude that mechanical loading of the tibia is anabolic for cortical bone in young and middle-aged female BALB/c mice.     

Recombinant human granulocyte colony-stimulating factor improves the compromised state of recipient mice without affecting the induction of specific tolerance in the cyclophosphamide-induced tolerance system.

The effects of recombinant human granulocyte CSF (rhG-CSF) on cyclophosphamide (CP)-induced tolerance was studied. In the recipient C57BL/10 Sn Slc (B10) mice given 1 x 10(8) B10.BR Sg Sn Slc (B10.BR) spleen cells (SC) on day -2 followed by 200 mg/kg CP on day 0, the number of leukocytes and neutrophils in the periphery declined to their minimum levels on day 4. When rhG-CSF in a dose of 200 micrograms/kg was given daily for 5 days to the B10 mice, which had been treated with B10.BR SC and CP, starting one day after the administration of CP, the leukocyte and neutrophil counts declined to the same levels as those in the B10 mice treated with B10.BR SC and CP alone on day 2. On day 4, however, the counts recovered to their normal levels. The nucleated cell count of the spleen in the B10 mice given B10.BR SC and CP followed by rhG-CSF decreased less and recovered faster than that in the B10 mice given B10.BR SC and CP. The case was found to be the same in bone marrow, and the difference did not reach statistical significance. When the recipient mice were inoculated i.p. with 4 x 10(4) Pseudomonas aeruginosa (GNB-139) on day 4, the survival of the B10 mice treated with B10.BR SC and CP was markedly improved by rhG-CSF administration. The administration of rhG-CSF did not affect either the prolongation or the specificity of skin allograft survival, as shown in an H-2 mis-matched combination of B10.BR----B10 and in an H-2 identical combination of AKR/J Sea(AKR)----C3H/HeN Crj (C3H). The tolerant state, which was demonstrated by various immune responses, such as CTL, delayed footpad reaction, and antibody, was also not affected by rhG-CSF. Furthermore, the basic mechanisms for inducing a long-lasting skin allograft tolerance in this system--namely, the specific destruction of Ag-stimulated and then proliferating mature T cells in the periphery, the establishment of mixed chimerism, and the intrathymic clonal deletion of immature T cells--were preserved even when rhG-CSF was given to C3H mice previously made tolerant of AKR.     

Characterization of Ath29, a major mouse atherosclerosis susceptibility locus, and identification of Rcn2 as a novel regulator of cytokine expression

Ath29 is an atherosclerosis susceptibility locus on chromosome 9 identified in an intercross between C57BL/6 (B6) and C3H/HeJ (C3H) apolipoprotein E-deficient (apoE(-/-)) mice. This locus was subsequently replicated in two separate intercrosses that developed early or advanced atherosclerotic lesions. The objective of this study was to characterize Ath29 through construction and analysis of a congenic strain and identify underlying candidate genes. A congenic line was constructed by introgressing the chromosomal segment harboring Ath29 from C3H.apoE(-/-) into B6.apoE(-/-) mice. Congenic mice developed significantly smaller early and advance atherosclerotic lesions than B6.apoE(-/-) mice. Microarray analysis revealed 317 genes to be differentially expressed in the aorta of congenic mice compared with B6.apoE(-/-) mice. Pathway analysis of these genes suggested the Ca(2+) signaling pathway to be implicated in regulating atherosclerosis susceptibility. Rcn2 is located underneath the linkage peak of Ath29 and involved in Ca(2+) signaling. Multiple single-nucleotide polymorphisms between B6 and C3H mice were detected within and surrounding Rcn2 with one single-nucleotide polymorphism falling within an upstream cAMP response element. Immunostaining demonstrated its expression in atherosclerotic lesions. Knockdown of Rcn2 with small interfering RNAs resulted in significant reductions in both baseline and oxidized phospholipid-induced VCAM-1 and monocyte chemoattractant protein-1 expression by endothelial cells. Ath29 is confirmed to be a major atherosclerosis susceptibility locus affecting both early and advanced lesion formation in mice, and Rcn2 is identified as a novel regulator of cytokine expression.     

Stimulation of Bone Formation in Cortical Bone of Mice Treated with a Receptor Activator of Nuclear Factor-κB Ligand (RANKL)-binding Peptide That Possesses Osteoclastogenesis Inhibitory Activity

To date, parathyroid hormone is the only clinically available bone anabolic drug. The major difficulty in the development of such drugs is the lack of clarification of the mechanisms regulating osteoblast differentiation and bone formation. Here, we report a peptide (W9) known to abrogate osteoclast differentiation in vivo via blocking receptor activator of nuclear factor-κB ligand (RANKL)-RANK signaling that we surprisingly found exhibits a bone anabolic effect in vivo. Subcutaneous administration of W9 three times/day for 5 days significantly augmented bone mineral density in mouse cortical bone. Histomorphometric analysis showed a decrease in osteoclastogenesis in the distal femoral metaphysis and a significant increase in bone formation in the femoral diaphysis. Our findings suggest that W9 exerts bone anabolic activity. To clarify the mechanisms involved in this activity, we investigated the effects of W9 on osteoblast differentiation/mineralization in MC3T3-E1 (E1) cells. W9 markedly increased alkaline phosphatase (a marker enzyme of osteoblasts) activity and mineralization as shown by alizarin red staining. Gene expression of several osteogenesis-related factors was increased in W9-treated E1 cells. Addition of W9 activated p38 MAPK and Smad1/5/8 in E1 cells, and W9 showed osteogenesis stimulatory activity synergistically with BMP-2 in vitro and ectopic bone formation. Knockdown of RANKL expression in E1 cells reduced the effect of W9. Furthermore, W9 showed a weak effect on RANKL-deficient osteoblasts in alkaline phosphatase assay. Taken together, our findings suggest that this peptide may be useful for the treatment of bone diseases, and W9 achieves its bone anabolic activity through RANKL on osteoblasts accompanied by production of several autocrine factors.     

The skeletal responsiveness to mechanical loading is enhanced in mice with a null mutation in estrogen receptor-beta

Mechanical loading caused by physical activity can stimulate bone formation and strengthen the skeleton. Estrogen receptors (ERs) play some role in the signaling cascade that is initiated in bone cells after a mechanical load is applied. We hypothesized that one of the ERs, ER-beta, influences the responsiveness of bone to mechanical loads. To test our hypothesis, 16-wk-old male and female mice with null mutations in ER-beta (ER-beta(-/-)) had their right forelimbs subjected to short daily loading bouts. The loading technique used has been shown to increase bone formation in the ulna. Each loading bout consisted of 60 compressive loads within 30 s applied daily for 3 consecutive days. Bone formation was measured by first giving standard fluorochrome bone labels 1 and 6 days after loading and using quantitative histomorphometry to assess bone sections from the midshaft of the ulna. The left nonloaded ulna served as an internal control for the effects of loading. Mechanical loading increased bone formation rate at the periosteal bone surface of the mid-ulna in both ER-beta(-/-) and wild-type (WT) mice. The ulnar responsiveness to loading was similar in male ER-beta(-/-) vs. WT mice, but for female mice bone formation was stimulated more effectively in ER-beta(-/-) mice (P < 0.001). We conclude that estrogen signaling through ER-beta suppresses the mechanical loading response on the periosteal surface of long bones.