Biomedical subjects

David J Baylink

Publications and source records attributed to David J Baylink.

At least 19 recordsLinked to original sources

PubMed2006

An improved mouse Sca-1+ cell-based bone marrow transplantation model for use in gene- and cell-based therapeutic studies.

This study sought to develop a murine bone marrow transplantation strategy that would yield consistently high levels of long-term engraftment without significant morbidity and mortality. Hematopoietic stem cell (HSC)-enriched Sca-1+ cells were used for transplantation because of their propensity of homing to bone marrow. Green fluorescent protein (GFP)-expressing transgenic mice were used as donors. Murine Sca-1+ cells were enriched 13-fold from whole bone marrow with immunomagnetic column chromatography. Retroorbital injections yielded highly reproducible and higher levels of engraftment compared with tail vein injections. The combination of W41/W41 recipient mice and sublethal irradiation preconditioning produced long-term engraftment with minimal morbidity and mortality. A 24-hour delay between the sublethal irradiation and transplantation did not affect the efficiency and level of engraftment, but provided flexibility with respect to the timing of transplantation. Based on these findings, a mouse Sca-1+ cell-based strategy, involving the retroorbital injection of Sca-1+ cells into sublethally irradiated, myelosuppressed W41/W41 recipient mice within 24 h after irradiation, was developed. Transplantation of lentiviral vector-transduced wild-type Sca-1+ cells expressing GFP by this strategy led to consistently high levels of long-term engraftment. In summary, this murine Sca-1+ cell-based strategy could be used in studies of HSC-based gene or cell therapies.

Animals↗

PubMed2006

Pregnancy-associated plasma protein-A increases osteoblast proliferation in vitro and bone formation in vivo.

Pregnancy-associated plasma protein (PAPP)-A, a protease for IGF binding protein (IGFBP)-2, -4, and -5, may enhance IGF action by increasing its bioavailability. Here we have determined the role and mechanism of action of PAPP-A in the regulation of osteoblast proliferation in vitro and bone metabolism in vivo. Recombinant PAPP-A (100 ng/ml) significantly increased osteoblast proliferation and free IGF-I concentration. These effects were abolished by noncleavable IGFBP-4, suggesting that PAPP-A promotes osteoblast proliferation by increasing IGF bioavailability. To determine whether PAPP-A exerts an anabolic effect on bone in vivo, we developed transgenic mice that overexpress PAPP-A in osteoblasts using the 2.3-kb rat type I collagen promoter. Consistent with the increase in IGFBP-4 proteolysis, free IGF-I concentration was significantly increased in the conditioned medium of cultured osteoblasts derived from transgenic mice compared with the wild-type littermates. Calvarial bone thickness, bone marrow cavity, and skull bone mineral density were significantly increased in transgenic mice. Bone size-related parameters in femur and tibia such as total bone area and periosteal circumference as determined by peripheral quantitated computed tomography and histological analysis were significantly increased in transgenic mice. Bone formation rate and osteoid surface were increased by more than 2-fold, whereas bone resorbing surface was unaffected. These anabolic effects were sustained with aging. These findings provide strong evidence that PAPP-A acts as a potent anabolic factor in the regulation of bone formation. Thus, enhancing IGF bioavailability by PAPP-A can be a powerful strategy in the treatment of certain metabolic diseases such as osteoporosis.

Animals↗

PubMed2006

Identification of genetic loci that regulate bone adaptive response to mechanical loading in C57BL/6J and C3H/HeJ mice intercross.

Strain-dependent differences in bone adaptive responses to loading among inbred mouse strains suggest that genetic background contributes significantly to adaptation to exercise. To explore the genetic regulation of response to loading, we performed a genome-wide search for linkage in a cross between two strains, a good responder, C57BL6/J (B6), and a poor responder, C3H/HeJ (C3H). Using a four-point bending model, the right tibia was loaded by applying 9 N force for 36 cycles for 12 days in 10-week-old female B6xC3H F2 mice. Changes in bone density (BMD) and bone size were evaluated in vivo by pQCT. Measurements from non-loaded left tibia were used as an internal control to calculate loading-induced percent increase in BMD and bone size, thus excluding the possibility of identifying background QTL(s) due to natural allelic variation in mapping strains. A genome-wide scan was performed using 111 microsatellite markers in DNA samples collected from 329 F2 mice. Heritability of bone adaptive response to loading was between 70 and 80%. The mean increase, expressed as percent of unloaded tibia, was 5% for BMD, 9% for periosteal circumference (PC), and 14% for cortical thickness in F2 mice (n = 329). All these phenotypes showed normal distributions. Absence of significant correlation between BMD response to four-point bending and body weight or bone size suggested that the bone adaptive response was independent of bone size. Interval mapping revealed that BMD response to four-point bending was influenced by three significant loci on Chrs 1 (log-of-odds ratio score (LOD) 3.4, 91.8 cM), 3 (LOD 3.6, 50.3 cM), and 8 (LOD 4.2, 60.1 cM) and one suggestive QTL on Chr 9 (LOD 2.5, 33.9 cM). Loading-induced increases in PC and Cth were influenced by four significant loci on Chrs 8 (LOD 3.0, 68.9 cM), 9 (LOD 3.0, 13.1 cM), 17 (LOD 3.0, 39.3 cM), and 18 (LOD 3.0, 0 cM) and two suggestive loci on Chr 9 (LOD 2.2, 24 cM) and 11 (LOD 2.1, 69.9 cM). Pairwise analysis showed the presence of several significant and suggestive interactions between loci on Chrs 1, 3, 8, and 13 for BMD trait. This is the first study that provides evidence for the presence of multiple genetic loci regulating bone anabolic responses to loading in the B6xC3H intercross. Knowledge of the genes underlying these loci could provide novel approaches to improve skeletal mass.

Adaptation, Biological↗

PubMed2006

An osteoclastic protein-tyrosine phosphatase is a potential positive regulator of the c-Src protein-tyrosine kinase activity: a mediator of osteoclast activity.

This study tested the hypothesis that an osteoclastic protein-tyrosine phosphatase, PTP-oc, enhances osteoclast activity through c-Src activation. The effects of several resorption activators and inhibitors on PTP-oc expression, resorption activity, and c-Src activation were determined in rabbit osteoclasts. PTP-oc expression was assayed with immunoblots and semi-quantitative RT-PCR. Osteoclastic activity was determined by the resorption pit assay; and c-Src activation was monitored by P-tyr527 (PY527) dephosphorylation, and in vitro kinase assay. Treatment of osteoclasts with PTH, PGE2, 1,25(OH)2D3, IL-1, but not RANKL or IL-6, significantly stimulated resorption activity, increased PTP-oc mRNA and protein levels, and reduced c-Src PY527 level with corresponding activation of c-Src protein-tyrosine kinase activity. The PTP-oc antisense phosphorothioated oligo treatment blocked the basal and IL-1alpha-mediated, but not RANKL-mediated, resorption activity of isolated osteoclasts. The antisense oligo treatment also significantly reduced the average depth of resorption pits created by rabbit osteoclasts under basal conditions. Calcitonin and alendondrate, significantly reduced resorption activity and PTP-oc expression, and increased c-Src PY527 with corresponding reduction in its PTK activity. The cellular PTP-oc protein level correlated with the resorption activity. Among the various signaling proteins co-immunoprecipitated with PTP-oc, the resorption effectors caused corresponding changes in the tyrosyl phosphorylation level of only c-Src. The GST-PTP-oc fusion protein dephosphorylated PY-527-containing c-Src peptide in time- and dose-dependent manner in vitro. In summary, (1) PTP-oc is regulated in part at transcriptional level, (2) upregulation of PTP-oc in osteoclasts led to c-Src activation, and (3) PY527 of c-Src may be a cellular substrate of PTP-oc. These findings are consistent with the hypothesis that PTP-oc is a positive regulator of c-Src in osteoclasts.

Animals↗

PubMed2006

Whole genome microarray analysis of growth hormone-induced gene expression in bone: T-box3, a novel transcription factor, regulates osteoblast proliferation.

Growth hormone (GH) is important in the development and maintenance of bone; however, the IGF-dependent and -independent molecular pathways involved remain to be established. We used microarray analysis to evaluate GH signaling pathways in 4-wk-old GH-deficient mice following a single injection of GH (4 mg/kg body wt) or PBS (n = 6/group) at 6 or 24 h after treatment. Six thousand one hundred sixty genes were differentially expressed at P </= 0.05, and 17% of these genes were identified at both time points. Several of the genes differentially expressed were expressed sequence tags, and the remaining genes fell into 49 Gene Ontology categories. For subsequent studies, we focused on T-box (Tbx)3, a novel transcription factor, which increased more than twofold at both time points. Real-time RT-PCR analysis determined that pretreatment with IGF-binding protein-4 did not block GH-induced Tbx3 expression in vitro. Pretreatment with TNF-alpha blocked GH-induced Tbx3 expression. Tbx3 expression increased during osteoblast differentiation and following BMP-7 and Wnt3a treatment (P </= 0.05). Blocking Tbx3 expression by small interfering RNA decreased cell number and [(3)H]Thymidine incorporation (P < 0.01). In conclusion, 1) GH caused acute changes in several novel genes, suggesting that many GH-induced signaling pathways and target genes remain to be discovered; 2) because Tbx3 expression is regulated in osteoblasts and blockage of Tbx3 expression decreased cell number and DNA synthesis, we propose that Tbx3 is an important determinant of osteoblast cell number.

Animals↗

PubMed2006

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.

Animals↗

PubMed2006

Microarray analysis of gene expression during the inflammation and endochondral bone formation stages of rat femur fracture repair.

Microarray analysis of gene expression was performed in the healing femur fractures of 13-week-old male rats during the inflammatory stage of repair, at 3 days post-fracture, and the endochondral bone formation stage of repair, at 11 days post-fracture. Multiple replicate pairs of fracture tissues paired with unfractured tissues, and unfractured control bones that had the stabilizing K-wire were introduced. This approach normalized the marrow contributions to the RNA repertoire. We identified 6555 genes with significant changes in expression in fracture tissues at 3 days and 11 days healing. The repertoire of growth factor genes expressed was also surprisingly restricted at both post-fracture intervals. The large number of Expressed Sequence Tags (ESTs) expressed at both post-fracture times indicates that several molecular pathways yet to be identified regulate fracture repair. The number of genes expressed during immune responses and inflammatory processes was restricted with higher expression largely during the early post-fracture analysis. Several of the genes identified in this study have been associated with regulation of cell and extracellular matrix interactions during scarless healing of fetal skin wounds. These observations suggest that these genes might also regulate the scarless healing characteristic of bone regeneration by similar mechanisms.

Animals↗

PubMed2006

Femur mechanical properties in the F2 progeny of an NZB/B1NJ x RF/J cross are regulated predominantly by genetic loci that regulate bone geometry.

UNLABELLED: Genetic analysis of an NZB/B1NJ x RF/J cross has identified QTLs for femur mechanical, geometric, and densitometric phenotypes. Most mechanical QTLs were associated with geometric QTLs, strongly suggesting common genetic regulation. INTRODUCTION: Previous studies have shown that bone architecture and BMD are important factors affecting bone strength, and both are genetically regulated. We conducted genetic analyses for loci regulating femur mechanical properties, geometric properties, and BMD in a cohort of F2 mice derived from intercross matings of (NZB/B1NJ x RF/J)F1 parents. MATERIALS AND METHODS: Femurs were isolated from 662 10-week-old females. Mechanical properties were determined for a femur from each animal by three-point bending. Geometric properties and volumetric BMD (vBMD) were determined by pQCT. Genotype data were obtained by PCR assays for polymorphic markers carried in the genomic DNA of each mouse. Genome-wide scans were carried out for co-segregation of genetic marker data with values from 23 different phenotypes. Quantitative trait loci (QTLs) were identified for mechanical, geometric, and mineral density phenotypes. RESULTS: QTLs for many phenotypes were significantly refined by covariate analyses using body weight and femur length. Major QTLs for mechanical and geometric phenotypes were found on chromosomes 5, 7, 9, 11, and 12. Nine chromosomal locations were identified with mechanical QTLs and 17 locations with one or more geometric QTLs. The significance of five mechanical and nine geometric QTLs was affected by the inclusion of covariates. These changes included both decreases and increases in significance. The QTLs on chromosomes 5 and 12 were decreased by inclusion of the covariates in the analysis, but QTLs on 7 and 11 were unaffected. Mechanical QTLs were almost always associated with geometric QTLs and less commonly (two of six) with vBMD QTLs. CONCLUSIONS: Genetic regulation of mechanical properties in the F(2) mice of this NZB/B1NJ x RF/J cross seems to be caused by genes regulating femur geometry.

Animals↗

PubMed2005

Mapping the dominant wound healing and soft tissue regeneration QTL in MRL x CAST.

We have used a mouse ear punch model and the QTL (quantitative trait loci) mapping technique to identify genes that are responsible for soft tissue regeneration. In the early studies, we have identified several QTL and have shown that the inheritance of ear healing was additive in one cross (MRL x SJL), and recessive in another cross (DBA x 129). Because CAST mice are genetically distinct and have a different genetic background, CAST would facilitate the identification of common and novel QTL when crossed with common inbred lines. We made a cross between super healer MRL and poor healer CAST and collected ear punch phenotype and marker genotype data from F(2). Ear punch healing exhibited a dominant mode of inheritance in this cross. There were three main QTL on Chromosomes 4, 9, and 17, and two suggestive QTL on Chromosomes 1 (new) and 7. Taken together, these QTL accounted for about 29% of total F2 variance of MRL x CAST. Compared with another study using the same cross, we found a totally different set of QTL. Two QTL interactions were identified by a full QTL model: Chromosomes 4 x 17 and 9 x 17; the latter reached to a statistical level at p < 0.05. These interactions explained about 4% of the F2 phenotypic variance. We conclude that soft tissue regeneration is controlled by multiple genes and locus vs. locus interactions.

Animals↗

PubMed2005

Identification and characterization of novel IGFBP5 interacting protein: evidence IGFBP5-IP is a potential regulator of osteoblast cell proliferation.

Insulin-like growth factor binding protein-5 (IGFBP5) is a multifunctional protein, which acts not only as a traditional binding protein, but also functions as a growth factor independent of IGFs to stimulate bone formation. It has been predicted that the intrinsic growth factor action of IGFBP5 involves binding of IGFBP5 to a putative receptor to induce downstream signaling pathways and/or nuclear translocation of IGFBP5 to influence transcription of genes involved in osteoblast cell proliferation/differentiation. Our study indentified proteins that bound to IGFBP5 using IGFBP5 as bait in a yeast two-hybrid screen of the U2 human osteosarcoma cell cDNA library. One of the clones that interacted strongly with the bait under high-stringency conditions corresponded to a novel IGFBP5 interacting protein (IGFBP5-IP) encoded by a gene that resides in mouse chromosome 10. The interaction between IGFBP5-IP and IGFBP5 is confirmed by in vitro coimmunoprecipitation studies that used pFlag and IGFBP5 polyclonal antibody, and cell lysates overexpressing both IGFBP5-IP and IGFBP5. Northern blot and RT-PCR analysis showed that the IGFBP-IP is expressed in both untransformed normal human osteoblasts and in osteosarcoma cell lines, which are known to produce IGFBP5. To determine the roles of IGFBP5-IP, we evaluated the effect of blocking the expression of IGFBP5-IP on osteoblast proliferation. We found that using a IGFBP5-IP-specific small interfering-hairpin plasmid resulted in a decrease in both basal and IGFBP5-induced osteoblast cell proliferation. On the basis of these findings, we predict that IGFBP5-IP may act as intracellular mediator of growth promoting actions of IGFBP5 and perhaps other osteoregulatory agents in bone cells.

Amino Acid Sequence↗

PubMed2005

Identification of quantitative trait loci that regulate obesity and serum lipid levels in MRL/MpJ x SJL/J inbred mice.

The total body fat mass and serum concentration of total cholesterol, HDL cholesterol, and triglyceride (TG) differ between standard diet-fed female inbred mouse strains MRL/MpJ (MRL) and SJL/J (SJL) by 38-120% (P < 0.01). To investigate genetic regulation of obesity and serum lipid levels, we performed a genome-wide linkage analysis in 621 MRLx SJL F2 female mice. Fat mass was affected by two significant loci, D11Mit36 [43.7 cM, logarithm of the odds ratio (LOD) 11.2] and D16Mit51 (50.3 cM, LOD 3.9), and one suggestive locus at D7Mit44 (50 cM, LOD 2.4). TG levels were affected by two novel loci at D1Mit43 (76 cM, LOD 3.8) and D12Mit201 (26 cM, LOD 4.1), and two suggestive loci on chromosomes 5 and 17. HDL and cholesterol concentrations were influenced by significant loci on chromosomes 1, 3, 5, 7, and 17 that were in the regions identified earlier for other strains of mice, except for a suggestive locus on chromosome 14 that was specific to the MRL x SJL cross. In summary, linkage analysis in MRL x SJL F2 mice disclosed novel loci affecting TG, HDL, and fat mass, a measure of obesity. Knowledge of the genes in these quantitative trait loci will enhance our understanding of obesity and lipid metabolism.

Adiposity↗

PubMed2005

Multiple genetic loci from CAST/EiJ chromosome 1 affect vBMD either positively or negatively in a C57BL/6J background.

UNLABELLED: Skeletal phenotype analyses of 10 B6.CAST-1 congenic sublines of mice have revealed evidence for the presence of three closely linked QTLs in Chr 1 that influence femoral vBMD both positively and negatively. INTRODUCTION: BMD is an important component of bone strength and a recognized predictor of risk for osteoporotic fracture. Our goal in this study was to fine map the chromosomal location of volumetric BMD (vBMD) quantitative trait loci (QTLs) in mouse distal chromosome 1 (Chr 1). MATERIALS AND METHODS: After several backcrosses of the B6.CAST-1T congenic strain, which carried the initial BMD QTL in Chr 1 with B6 mice, the N10F1 generation mice were intercrossed to obtain recombinations that yielded different regions of the QTL. Thirty-eight polymorphic markers were used to fine map the initial 1T QTL region (100-192 Mb). Different skeletal parameters were compared between the 10 sublines and B6 female mice at 16 weeks of age. A t-test was used to determine the significant difference between sublines and B6 control mice, whereas one-way ANOVA and posthoc (Newman-Keuls) tests were performed to compare the phenotype between the sublines. RESULTS: Significantly higher femur vBMD was found in sublines that carried cast alleles from 100 to 169 and 172 to 185 Mb of the centromere compared with the B6 control mice (10-12%, p < 0.001). However, sublines that carried cast alleles from 185 to 192 Mb showed significantly lower femur vBMD compared with the control mice (-6%, p < 0.05). Furthermore, femur vBMD phenotype showed a negative correlation with endosteal circumference (r = -0.8, p = 0.003), and a strong correlation with cortical thickness for combined data from the 10 sublines (r = 0.97, p < 0.001). Moreover, a high correlation was found between body weight and both periosteal and endosteal circumferences for sublines carrying cast alleles from 167 to 175, 168 to 185, and 169 to 185 Mb, whereas no significant correlation was found between these parameters for sublines carrying cast alleles from 172 to 185 Mb. CONCLUSIONS: Genetic analysis using congenic sublines revealed that the initial BMD QTL on Chr 1 is a complex site with multiple loci affecting bone phenotypes, showing the value of the congenic approach in clearly identifying loci that control specific traits.

Alleles↗

PubMed2005

New quantitative trait loci that regulate wound healing in an intercross progeny from DBA/1J and 129 x 1/SvJ inbred strains of mice.

Wound healing/regeneration mouse models are few, and studies performed have mainly utilized crosses between MRL/MPJ (a good healer) and SJL/J (a poor healer) or MRL/lpr (a good healer) and C57BL/6J (a poor healer). Wound healing is a complex trait with many genes involved in the expression of the phenotype. Based on data from previous studies that common and additional quantitative trait loci (QTL) were identified using different crosses of inbred strains of mice for various complex traits, we hypothesized that a new cross would identify common and additional QTL, unique modes of inheritance, and interacting loci, which are responsible for variation in susceptibility to fast wound healing. In this study, we crossed DBA/1J (DBA, a good healer) and 129/SvJ (129, a poor healer) and performed a genome-wide scan using 492 (DBA x 129) F2 mice and 98 markers to identify QTL that regulate wound healing/regeneration. Four QTL on chromosomes 1, 4, 12, and 18 were identified which contributed toward wound healing in F2 mice and accounted for 17.1% of the phenotypic variation in ear punch healing. Surprisingly, locus interactions contributed to 55.7% of the phenotype variation in ear punch healing. In conclusion, we have identified novel QTL and shown that minor interacting loci contribute significantly to wound healing in DBA x 129 mice cross.

Animals↗

PubMed2005

Pregnancy-associated plasma protein-A regulates myoblast proliferation and differentiation through an insulin-like growth factor-dependent mechanism.

Pregnancy-associated plasma protein-A (PAPP-A), a member of the metalloproteinase superfamily, is an important regulator of mammalian growth and development. However, the role of PAPP-A and its mechanism of action in various cellular processes remain unknown. In this study, we have investigated the role of PAPP-A in skeletal myogenesis using C2C12 myoblasts. Recombinant PAPP-A was purified from the conditioned medium of HT1080 cells overexpressing PAPP-A. Treatment of C2C12 myoblasts with PAPP-A increased their proliferation in a dose- and time-dependent manner. Addition of exogenous PAPP-A also increased the myotube formation and the activity of creatine kinase in C2C12 cultures. Transient overexpression of the full-length PAPP-A-(1-1547), but not truncated protease-inactive N-terminal PAPP-A-(1-920) or C-terminal PAPP-A-(1100-1547), significantly enhanced the proliferation of C2C12 myoblasts. In vitro and in situ experiments demonstrated that PAPP-A cleaves insulin-like growth factor-binding protein (IGFBP)-2, but not IGFBP-3, in the conditioned medium of C2C12 myoblasts. Overexpression of PAPP-A led to degradation of the IGFBP-2 produced by C2C12 myoblasts and increased free IGF-I concentrations without affecting total IGF-I concentrations. Addition of protease-resistant IGFBP-4 completely abolished the PAPP-A-induced proliferation of C2C12 myoblasts. Our results demonstrate that 1) PAPP-A increases the proliferation and differentiation of myoblasts, 2) the stimulatory effect of PAPP-A on myogenesis is governed by its proteolytic activity, and 3) PAPP-A promotes skeletal myogenesis by increasing the amount of free IGFs via specific degradation of IGFBP-2 produced by myoblasts.

Animals↗

PubMed2005

Mechanical loading-induced gene expression and BMD changes are different in two inbred mouse strains.

Our goal is to evaluate skeletal anabolic response to mechanical loading in different age groups of C57B1/6J (B6) and C3H/HeJ (C3H) mice with variable loads using bone size, bone mineral density (BMD), and gene expression changes as end points. Loads of 6-9 N were applied at 2 Hz for 36 cycles for 12 days on the tibia of 10-wk-old female B6 and C3H mice. Effects of a 9-N load on 10-, 16-, and 36-wk-old C3H mice were also studied. Changes in bone parameters were measured using peripheral quantitative computed tomography, and gene expression was determined by real-time PCR. Total volumetric BMD was increased by 5 and 15%, respectively, with 8- and 9-N loads in the B6, but not the C3H, mice. Increases of 20 and 12% in periosteal circumference were reflected by dramatic 44 and 26% increases in total area in B6 and C3H mice, respectively. The bone response to bending showed no difference in the three age groups of B6 and C3H mice. At 2 days, mechanical loading resulted in significant downregulation in expression of bone resorption (BR), but not bone formation (BF) marker genes. At 4 and 8 days of loading, expression of BF marker genes (type I collagen, alkaline phosphatase, osteocalcin, and bone sialoprotein) was increased two- to threefold and expression of BR marker genes (matrix metalloproteinase-9 and thrombin receptor-activating peptide) was decreased two- to fivefold. Although expression of BF marker genes was upregulated four- to eightfold at 12 days of training, expression of BR marker genes was upregulated seven- to ninefold. Four-point bending caused significantly greater changes in expression of BF and BR marker genes in bones of the B6 than the C3H mice. We conclude that mechanical loading-induced molecular pathways are activated to a greater extent in the B6 than in the C3H mice, resulting in a higher anabolic response in the B6 mice.

Alkaline Phosphatase↗

PubMed2005

Isolation of wound healing/regeneration genes using restrictive fragment differential display-PCR in MRL/MPJ and C57BL/6 mice.

Wound healing in mammals can take several weeks to months and the process is always accompanied by scar formation. Wound healing mechanisms that mimic regeneration are not found in most mature mammalian tissues. However, the MRL/MPJ (MRL) mouse has the unique capacity to regenerate ear hole wound completely in less than a month. To identify genes involved in wound healing without a scar, we chose to use restriction fragment differential display-PCR to isolate genes differentially expressed in the MRL (good healer) mouse and the C57BL/6 (poor healer) mouse at different stages of wound healing. We identified 36 genes that were differentially expressed in the regenerating tissue of good and poor healer strains of which several genes are also genetically linked to wound healing and thus are potential candidate genes for scarless wound healing.

Animals↗

PubMed2005

Spontaneous fractures in the mouse mutant sfx are caused by deletion of the gulonolactone oxidase gene, causing vitamin C deficiency.

UNLABELLED: Using a mouse mutant that fractures spontaneously and dies at a very young age, we identified that a deletion of the GULO gene, which is involved in the synthesis of vitamin C, is the cause of impaired osteoblast differentiation, reduced bone formation, and development of spontaneous fractures. INTRODUCTION: A major public health problem worldwide, osteoporosis is a disease characterized by inadequate bone mass necessary for mechanical support, resulting in bone fracture. To identify the genetic basis for osteoporotic fractures, we used a mouse model that develops spontaneous fractures (sfx) at a very early age. MATERIALS AND METHODS: Skeletal phenotype of the sfx phenotype was evaluated by DXA using PIXImus instrumentation and by dynamic histomorphometry. The sfx gene was identified using various molecular genetic approaches, including fine mapping and sequencing of candidate genes, whole genome microarray, and PCR amplification of candidate genes using cDNA and genomic DNA as templates. Gene expression of selected candidate genes was performed using real-time PCR analysis. Osteoblast differentiation was measured by bone marrow stromal cell nodule assay. RESULTS: Femur and tibial BMD were reduced by 27% and 36%, respectively, in sfx mice at 5 weeks of age. Histomorphometric analyses of bones from sfx mice revealed that bone formation rate is reduced by >90% and is caused by impairment of differentiated functions of osteoblasts. The sfx gene was fine mapped to a 2 MB region containing approximately 30 genes in chromosome 14. By using various molecular genetic approaches, we identified that deletion of the gulonolactone oxidase (GULO) gene, which is involved in the synthesis of ascorbic acid, is responsible for the sfx phenotype. We established that ascorbic acid deficiency caused by deletion of the GULO gene (38,146-bp region) contributes to fractures and premature death because the sfx phenotype can be corrected in vivo by treating sfx mice with ascorbic acid and because osteoblasts derived from sfx mice are only able to form mineralized nodules when treated with ascorbic acid. Treatment of bone marrow stromal cells derived from sfx/sfx mice in vitro with ascorbic acid increased expression levels of type I collagen, alkaline phosphatase, and osteocalcin several-fold. CONCLUSION: The sfx is a mutation of the GULO gene, which leads to ascorbic acid deficiency, impaired osteoblast cell function, and fractures in affected mice. Based on these and other findings, we propose that ascorbic acid is essential for the maintenance of differentiated functions of osteoblasts and other cell types.

Animals↗

PubMed2005

Bayesian shrinkage estimation of quantitative trait loci parameters.

Mapping multiple QTL is a typical problem of variable selection in an oversaturated model because the potential number of QTL can be substantially larger than the sample size. Currently, model selection is still the most effective approach to mapping multiple QTL, although further research is needed. An alternative approach to analyzing an oversaturated model is the shrinkage estimation in which all candidate variables are included in the model but their estimated effects are forced to shrink toward zero. In contrast to the usual shrinkage estimation where all model effects are shrunk by the same factor, we develop a Bayesian method that allows the shrinkage factor to vary across different effects. The new shrinkage method forces marker intervals that contain no QTL to have estimated effects close to zero whereas intervals containing notable QTL have estimated effects subject to virtually no shrinkage. We demonstrate the method using both simulated and real data for QTL mapping. A simulation experiment with 500 backcross (BC) individuals showed that the method can localize closely linked QTL and QTL with effects as small as 1% of the phenotypic variance of the trait. The method was also used to map QTL responsible for wound healing in a family of a (MRL/MPJ x SJL/J) cross with 633 F(2) mice derived from two inbred lines.

Animals↗