Our laboratory is investigating two complex traits--ovarian tumorigenesis and acquisition of skeletal properties--through genetic and biologic inquiries using mouse models. For the juvenile ovarian granulosa cell tumor investigation, we discovered two linked Chromosome X loci that interact to promote ovarian granulosa cell tumor frequency. Sequencing of specific candidate genes for both loci are in progress to begin analyses of coding regions. Additional genetic analyses showed several modifier loci supporting an established tumor-promoting locus on Chromosome 4. In companion dietary intervention studies, we are investigating how natural phytoestrogens, compounds found in plants such as cabbage and beans, interact with the body's own hormones and affect spontaneous tumor initiation frequency. Finally, biologic and genetic factors driving the malignant progression of this cancer model are under investigation.
As we investigate the complex process of acquiring skeletal bone mineral density, we have positionally cloned a defined region on distal mouse Chromosome 1 that carries three linked genes that regulate cortical or trabecular bone in females, males, or both sexes. Similar work with Chromosome 4 indicates the same complexity for bone compartments and sexes. These regions are of translational interest as both share homologous sets of linked genes with human Chromosome 1q and 1p, with associated adult human skeletal regulation. These investigations represent a major current effort to associate candidate genes with the regulatory biochemical mechanisms in bone.
Complex trait analyses: Adult skeletal acquisition and ovarian tumorigenesis
Adult skeletal acquisition
The heritability of skeletal bone mineral density (BMD) in humans has been recognized for several decades, yet even locating the chromosomal regions carrying that genetic regulation continues to be a formidable challenge. Our group and outside collaborators have pursued this complex trait in mice as an experimental system for phenotype decomposition, gene identification, and functional attributes of those quantitative trait loci (QTL). Density, size, microstructure and biomechanical strength are examples of phenotypes that differ among inbred strains of mice. Our particular strains of interest are C57BL/6J (B6, low volumetric BMD), C3H/HeJ (C3H, high volumetric BMD), and CAST/EiJ (CAST, high volumetric BMD), all of which exhibit many allelic differences regulating normal bone.
Positional cloning efforts for volumetric bone mineral density (vBMD) have concentrated on mouse Chromosomes (Chrs) 1, 4, and 18 that account for the largest variation in vBMD between B6 and C3H. Nested congenic sublines carrying 1-8 Mb-sized C3H donor segments on distal Chr 1 transferred to the B6 background have been measured for femoral bone parameters of vBMD and trabecular microstructure (by MicroCT40). The histomorphometric data revealed sex specific differences in cellular and bone formation parameters. We found that the original BMD QTL mapped to distal Chr 1 consists of 3 QTL with different effects on vBMD and trabecular bone in both sexes. Compared with B6 controls, femoral vBMD, mineral content and cortical thickness were significantly increased in congenic subline females, but not in males, carrying C3H alleles at QTL-1. Both females and males carrying C3H alleles at QTL-1 showed marked increases in bone volume fraction (BV/TV) compared to B6 mice. Females increased BV/TV by increasing trabecular thickness, whereas males increased trabecular number. In addition, the MicroCT40 data showed two unique QTL for male trabecular bone, QTL-2 and QTL-3, which may interact to regulate trabecular thickness and number. These QTL are closely linked with, and proximal to, QTL-1. According to the Ensembl Mouse genetic database, QTL-1 resides in a 0.14 Mb region containing 2 genes, QTL-2 resides in a 2.93 Mb region containing 71 genes, and QTL-3 resides in a 1.27 Mb region containing 31 genes. To date, gene expression studies with young adult bone have not yet yielded genotype and sex specific differential expression in congenic versus B6 progenitor that matches the measured bone phenotypes. Nevertheless, efforts continue to focus on QTL-1 and the osteoblast and osteoclast cell types for molecular genetic regulation that leads to enhanced bone parameters. Mice and humans share genetic homology between distal mouse Chr 1 and human chr 1q21-24 that is associated with adult human skeletal regulation. Gender and compartment specific regulatory QTL in the mouse suggest the need to partition human data by sex to improve accuracy of mapping and genetic loci identification.
Companion studies of nested congenics have also been conducted with Drs. B. Edderkaoui and S. Mohan, collaborators at J.L. Pettis VA Medical Center, Loma Linda, Calif. Using nested congenics, again on B6 background, but this time carrying distal Chr 1 segments from CAST, evidence for multiple vBMD QTL has been developed, which also display sex specific regulatory features. In addition, expression profiling of genes in the distal Chr 1 region and SNP analyses identified Duffy blood antigen (Dfy; also Darc) as a candidate gene for one BMD QTL. The involvement of Dfy protein in BMD variation was supported by the reduction in vBMD phenotype of Dfy-knockout mice. Thus, CAST alleles for Dfy may regulate BMD negatively via increasing osteoclast formation, whereas C3H alleles for Dfy did not show expression differences. Nevertheless, the genetic association between Dfy gene polymorphisms and BMD variations in humans merits investigation.
In summary, the distal Chr 1 region in mouse, with homology to chr 1q21-q24, harbors complex regulation for bone, with more genes likely to be discovered as these investigations proceed. Indeed, the nested congenic sets established for distal Chr 4 and Chr 18 are yielding much the same picture of rich genetic regulation.
Granulosa cell (GC) carcinoma of the ovarian follicles occurs in SWR-derived inbred strains of mice during pubertal maturation of the gonad. These spontaneous tumors are heritable, with initiation controlled by an SWR-derived oncogenic allele on distal Chr 4 that we are continuing to positionally clone using SWR.CAST congenics. Additional modifier loci are found on Chrs 1, 2, 9, 12, 13 and 15; two quantitative trait loci (QTL) are found on Chr X. Frequency of ovarian tumors ranges from less than 1 percent to 25 percent of females at 8 weeks of age, depending on their genetic background.
Tumorigenesis and frequency can be enhanced by exogenous or endogenous androgen and suppressed by exogenous estrogen. We sought to determine whether gonadotropic stimulation was sufficient to initiate GC tumors in a graft model system, and to determine the potential for dietary isoflavones (genistein and daidzein) as alternatives to E2 for tumor chemoprevention in vivo. Using a test system of tumor-susceptible ovaries engrafted to hosts that were both immune system and gonadotropin deficient (CB17;HPG-Prkdcscid Gnrh1hpg/Bm), we found that human chorionic gonadotropin (LH analog), but not ovine FSH, initiated GC tumorigenesis, suggesting that the LH surge at puberty initiates GC tumor development in genetically susceptible mice. The chemopreventive potential of phytoestrogens on GC tumor frequency was assessed with (SWRxSWXJ9)F1 females reared on an isoflavone-free diet versus isoflavone supplemented diet (daidzein and genistein). It was observed that (SWR x SWXJ9)F1 females reared on isoflavone-supplemented diet maintained significantly higher GC tumor frequency (22%) than females reared on isoflavone-free diet (11%), and that non-tumor bearing siblings reared on the isoflavones had significantly increased ovarian weight, indicative of an overall stimulation of the reproductive hormone axis.
The inability of isoflavones to prevent GC tumor initiation could be explained by the fact that gonadotropin levels were not reduced by the dietary isoflavone supplementation during the critical window for tumor initiation. This finding agrees with the genetic evidence that signaling via the ESR1 receptor is most important for negative feedback regulation of gonadotropin release, while pharmacological data shows that the isoflavones genistein and daidzein show preferential binding and trans-activation of the ESR2 receptor subtype. The ability of isoflavones to support GC tumor development in this animal model raises the question as to their mechanism of action. The genistein and daidzein supplemented diet did not support GC tumor initiation in genetically susceptible ovaries grafted into hpg/hpg, scid/scid recipients, suggesting that the isoflavones cannot stimulate GC tumor initiation independently from gonadotropic stimulation. The supportive action of the isoflavones for GC tumor initiation may be a function of their ESR2 receptor subtype specificity, acting via trans-activation of ESR2 to support granulosa cell differentiation and follicular development in vivo.
Co-Principal Investigators: Leah Rae Donahue, Ph.D., Clifford J. Rosen, M.D.
Research Assistant III: Kathryn L. Shultz, B.S.
Research Assistant II: Victoria DeMambro, B.S.
Research Assistant I: Harold F. Coombs, III, Krista M. Delahunty, B.S., M.A.
Executive Assistant: Aimée Picard
Beamer WG, Shultz KL, Coombs HF 3rd, Demambro VE, Reinholdt LG, Ackert-Bicknell CL, Canalis E, Rosen CJ, Donahue LR. BMD regulation on mouse distal chromosome 1, candidate genes, and response to ovariectomy or dietary fat. J Bone Miner Res. 2011 Jan;26(1):88-99. PubMed PMID: 20687154.
Canalis E, Smerdel-Ramoya A, Durant D, Economides AN, Beamer WG, Zanotti S. Nephroblastoma overexpressed (Nov) inactivation sensitizes osteoblasts to bone morphogenetic protein-2, but nov is dispensable for skeletal homeostasis. Endocrinology. 2010 Jan;151(1):221-33. Epub 2009 Nov 24. PubMed PMID: 19934377; PubMed Central PMCID: PMC2803142.
Canalis E, Zanotti S, Beamer WG, Economides AN, Smerdel-Ramoya A. Connective tissue growth factor is required for skeletal development and postnatal skeletal homeostasis in male mice. Endocrinology. 2010 Aug;151(8):3490-501. Epub 2010 Jun 9. PubMed PMID: 20534727; PubMed Central PMCID: PMC2940511.
DeMambro VE, Kawai M, Clemens TL, Fulzele K, Maynard JA, Marin de Evsikova C, Johnson KR, Canalis E, Beamer WG, Rosen CJ, Donahue LR. A novel spontaneous mutation of Irs1 in mice results in hyperinsulinemia, reduced growth, low bone mass and impaired adipogenesis. J Endocrinol. 2010 Mar;204(3):241-53. Epub 2009 Dec 23. PubMed PMID: 20032200.
Jiao F, Chiu H, Jiao Y, de Rijk WG, Li X, Eckstein EC, Beamer WG, Gu W. Quantitative trait loci for tibial bone strength in C57BL/6J and C3H/HeJ inbred strains of mice. J Genet. 2010 Apr;89(1):21-7. PubMed PMID: 20505243.
Delahunty KM, Horton LG, Coombs HF 3rd, Shultz KL, Svenson KL, Marion MA, Holick MF, Beamer WG, Rosen CJ. Gender- and compartment-specific bone loss in C57BL/6J mice: correlation to season? J Clin Densitom. 2009 Jan-Mar;12(1):89-94. PubMed PMID: 19195621.
Xiong Q, Jiao Y, Hasty KA, Canale ST, Stuart JM, Beamer WG, Deng HW, Baylink D, Gu W. Quantitative trait loci, genes, and polymorphisms that regulate bone mineral density in mouse. Genomics. 2009 May;93(5):401-14. Epub 2009 Jan 14. Review. PubMed PMID: 19150398; PubMed Central PMCID: PMC2901167.
Ackert-Bicknell CL, Demissie S, Mar√≠n de Evsikova C, Hsu YH, DeMambro VE, Karasik D, Cupples LA, Ordovas JM, Tucker KL, Cho K, Canalis E, Paigen B, Churchill GA, Forejt J, Beamer WG, Ferrari S, Bouxsein ML, Kiel DP, Rosen CJ. PPARG by dietary fat interaction influences bone mass in mice and humans. J Bone Miner Res. 2008 Sep;23(9):1398-408. PubMed PMID: 18707223; PubMed Central PMCID: PMC2683155.
DeMambro VE, Clemmons DR, Horton LG, Bouxsein ML, Wood TL, Beamer WG, Canalis E, Rosen CJ. Gender-specific changes in bone turnover and skeletal architecture in igfbp-2-null mice. Endocrinology. 2008 May;149(5):2051-61. Epub 2008 Feb 14. PubMed PMID: 18276763; PubMed Central PMCID: PMC2329262.
Ishimori N, Stylianou IM, Korstanje R, Marion MA, Li R, Donahue LR, Rosen CJ, Beamer WG, Paigen B, Churchill GA. Quantitative trait loci for BMD in an SM/J by NZB/BlNJ intercross population and identification of Trps1 as a probable candidate gene. J Bone Miner Res. 2008 Sep;23(9):1529-37. PubMed PMID: 18442308; PubMed Central PMCID: PMC2586053.
Xiong Q, Han C, Beamer WG, Gu W. A close examination of genes within quantitative trait loci of bone mineral density in whole mouse genome. Crit Rev Eukaryot Gene Expr. 2008;18(4):323-43. Review. PubMed PMID: 18652562.
Beamer WG, Shultz KL, Ackert-Bicknell CL, Horton LG, Delahunty KM, Coombs HF 3rd, Donahue LR, Canalis E, Rosen CJ. Genetic dissection of mouse distal chromosome 1 reveals three linked BMD QTLs with sex-dependent regulation of bone phenotypes. J Bone Miner Res. 2007 Aug;22(8):1187-96. PubMed PMID: 17451375.
Dorward AM, Shultz KL, Beamer WG. LH analog and dietary isoflavones support ovarian granulosa cell tumor development in a spontaneous mouse model. Endocr Relat Cancer. 2007 Jun;14(2):369-79. PubMed PMID: 17639051.
Edderkaoui B, Baylink DJ, Beamer WG, Shultz KL, Wergedal JE, Mohan S. Genetic regulation of femoral bone mineral density: complexity of sex effect in chromosome 1 revealed by congenic sublines of mice. Bone. 2007 Sep;41(3):340-5. Epub 2007 Jun 6. PubMed PMID: 17618849.
Edderkaoui B, Baylink DJ, Beamer WG, Wergedal JE, Porte R, Chaudhuri A, Mohan S. Identification of mouse Duffy antigen receptor for chemokines (Darc) as a BMD QTL gene. Genome Res. 2007 May;17(5):577-85. Epub 2007 Apr 6. PubMed PMID: 17416748; PubMed Central PMCID: PMC1855174.
Jiao Y, Chiu H, Fan Z, Jiao F, Eckstein EC, Beamer WG, Gu W. Quantitative trait loci that determine mouse tibial nanoindentation properties in an F2 population derived from C57BL/6J x C3H/HeJ. Calcif Tissue Int. 2007 Jun;80(6):383-90. Epub 2007 Jun 7. PubMed PMID: 17551771.
Ng AH, Wang SX, Turner CH, Beamer WG, Grynpas MD. Bone quality and bone strength in BXH recombinant inbred mice. Calcif Tissue Int. 2007 Sep;81(3):215-23. Epub 2007 Jul 20. PubMed PMID: 17638038.
Robling AG, Warden SJ, Shultz KL, Beamer WG, Turner CH. Genetic effects on bone mechanotransduction in congenic mice harboring bone size and strength quantitative trait loci. J Bone Miner Res. 2007 Jul;22(7):984-91. PubMed PMID: 17371164.
Yan J, Jiao Y, Jiao F, Stuart J, Donahue LR, Beamer WG, Li X, Roe BA, LeDoux MS, Gu W. Effects of carbonic anhydrase VIII deficiency on cerebellar gene expression profiles in the wdl mouse. Neurosci Lett. 2007 Feb 21;413(3):196-201. Epub 2006 Dec 15. PubMed PMID: 17174474; PubMed Central PMCID: PMC1865515.
Yu H, Mohan S, Edderkaoui B, Masinde GL, Davidson HM, Wergedal JE, Beamer WG, Baylink DJ. Detecting novel bone density and bone size quantitative trait loci using a cross of MRL/MpJ and CAST/EiJ inbred mice. Calcif Tissue Int. 2007 Feb;80(2):103-10. Epub 2007 Feb 2. PubMed PMID: 17308992.