Nuclear Receptors of the Adrenal Cortex: Genomic Targets of Steroidogenic Factor 1 and Evidence of Adrenal Failure in DAX1-Deficient Mice.

Steroidogenic Factor 1 (SF1) and DAX1 are nuclear receptors that play an integral role in the adrenal cortex. SF1 is a transcription factor that regulates expression of myriad genes, including those involved in steroidogenesis, production of various endocrine hormones, and development of the adrenals and gonads. DAX1 is an orphan nuclear receptor that inhibits SF1 and, when mutated, is responsible for X-linked Adrenal Hypoplasia Congenita (AHC). In the adrenal cortex, adrenocorticotropic hormone (ACTH) activates SF1-mediated transcription of the genes involved in cholesterol transport and steroid production. However, SF1 is also implicated in adrenal proliferation and differentiation; therefore, it is likely that several different signaling pathways regulate SF1-dependent transcription by altering cofactor availability or by feedback mechanisms that modulate signaling molecules that impinge on SF1 transactivation. This thesis describes three genomic targets of SF1 that provide feedback upon and affect subsequent SF1 transcriptional activity. First, I describe the identification of Edg5, the sphingosine 1-phosphate receptor, as a target of SF1 using chromatin immunoprecipitation-chip microarray (ChIP-chip). Activation of Edg5 may alter levels of intracellular sphingosine 1-phosphate, a ligand of SF1 that activates steroidogenesis and stimulates release of adrenal hormones. I also describe association of SF1 on the adenylyl cyclase 4 (Adcy4) promoter. Though usually an activator, SF1 represses transcription of Adcy4, which may alter cAMP-dependent activation of SF1. Finally, I describe an interaction between SF1 and GR to activate expression of Dax1, which is abrogated by ACTH. In all three cases, the genomic target of SF1 encodes a factor that can modulate SF1-dependent transcription. While loss of DAX1 in humans results in AHC, Dax1 deficient-mice have a mild adrenal phenotype. To reconcile these disparate phenotypes, I analyzed Dax1-deficient mice across their lifespan. Young Dax1-deficent mice have increased steroidogenic capacity, expression of steroidogenic enzymes, and proliferation. However, after 60 weeks all three measures are dramatically reduced, suggesting that loss of Dax1 may result in premature differentiation resulting in ultimate exhaustion of the adrenal cortex, which has implications for AHC. Overall, this dissertation contributes to the understanding of both SF1-dependent gene regulation and the significance of DAX1 in homeostasis of the adrenal cortex.Ph.D.Cellular & Molecular BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/78891/1/jscheys_1.pd

MTORC1 signaling and regulation of pancreatic β-cell mass

The capacity of β cells to expand in response to insulin resistance is a critical factor in the development of type 2 diabetes. Proliferation of β cells is a major component for these adaptive responses in animal models. The extracellular signals responsible for β-cell expansion include growth factors, such as insulin, and nutrients, such as glucose and amino acids. AKT activation is one of the important components linking growth signals to the regulation of β-cell expansion. Downstream of AKT, tuberous sclerosis complex 1 and 2 (TSC1/2) and mechanistic target of rapamycin complex 1 (mTORC1) signaling have emerged as prime candidates in this process, because they integrate signals from growth factors and nutrients. Recent studies demonstrate the importance of mTORC1 signaling in β cells. This review will discuss recent advances in the understanding of how this pathway regulates β-cell mass and present data on the role of TSC1 in modulation of β-cell mass. Herein, we also demonstrate that deletion of Tsc1 in pancreatic β cells results in improved glucose tolerance, hyperinsulinemia and expansion of β-cell mass that persists with aging

GREB1 is a critical regulator of hormone dependent breast cancer growth

Background Estrogen plays a central role in breast cancer pathogenesis and many potent risk factors for the development of the disease can be explained in terms of increased lifetime exposure to estrogen. Although estrogen regulated genes have been identified, those critically involved in growth regulation remain elusive.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44228/1/10549_2005_Article_1483.pd

Association of genetic variation in tamoxifen-metabolizing enzymes with overall survival and recurrence of disease in breast cancer patients

Tamoxifen has been a mainstay of adjuvant therapy for breast cancer for many years. We sought to determine if genetic variability in the tamoxifen metabolic pathway influenced overall survival in breast cancer patients treated with tamoxifen. We examined functional polymorphisms in CYP2D6, the P450 catalyzing the formation of active tamoxifen metabolites, and UGT2B15, a Phase II enzyme facilitating the elimination of active metabolite in a retrospective study of breast cancer patients. We also examined whether the combination of variant alleles in SULT1A1 and UGT2B15 had more of an impact on overall survival in tamoxifen-treated patients than when the genes were examined separately.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44227/1/10549_2004_Article_7751.pd

GREB1 is a novel androgen-regulated gene required for prostate cancer growth

BACKGROUND Gene regulated in breast cancer 1 (GREB1) is a novel estrogen-regulated gene shown to play a pivotal role in hormone-stimulated breast cancer growth. GREB1 is expressed in the prostate and its putative promoter contains potential androgen receptor (AR) response elements. METHODS We investigated the effects of androgens on GREB1 expression and its role in androgen-dependent prostate cancer growth. RESULTS Real-time PCR demonstrated high level GREB1 expression in benign prostatic hypertrophy (BPH), localized prostate cancer (L-PCa), and hormone refractory prostate cancer (HR-PCa). Androgen treatment of AR-positive prostate cancer cells induced dose-dependent GREB1 expression, which was blocked by anti-androgens. AR binding to the GREB1 promoter was confirmed by chromatin immunoprecipitation (ChIP) assays. Suppression of GREB1 by RNA interference blocked androgen-stimulated LNCaP cell proliferation. CONCLUSIONS GREB1 is expressed in proliferating prostatic tissue and prostate cancer, is regulated by androgens, and suppression of GREB1 blocks androgen-induced growth suggesting GREB1 may be critically involved in prostate cancer proliferation. Prostate © 2006 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/49275/1/20403_fta.pd

Mechanical Analysis of Extra-Articular Knee Ligaments. Part two: Tendon grafts used for knee ligament reconstruction

The aim of this study was to provide information about the mechanical properties of grafts used for knee ligament reconstructions and to compare those results with the mechanical properties of native knee ligaments.status: publishe

Genotyping for polymorphic drug metabolizing enzymes from paraffin-embedded and immunohistochemically stained tumor samples

Paraffin-embedded tumor samples are valuable in the study of cancer for routine staging, tumor marker analysis, and in retrospective studies to test new prognostic and predictive biomarkers. Their utility in retrospective pharmacogenetic analysis of clinical trials has yet to be evaluated. We set out to establish genotyping methods for relevant genes from archival tumor samples and determine if fixation, processing or somatic changes in the tumor might affect our ability to identify germ-line polymorphisms. To establish the assays, paraffin blocks were made using pellets prepared from eight tumor cell lines. DNA was isolated from viable cells and from sections from these blocks, and genotyped for polymorphisms in CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A5 and MDR1 using conventional PCR-RFLP assays. This demonstrated that fixation and processing did not alter the genotypes obtained (100% concordance). Next, sections were obtained from paraffin-embedded archival breast samples from 10 patients for whom gDNA isolated from peripheral blood was available for comparison. Concordance was complete with the same genotype being obtained for 100% of the samples tested. Attempts to extend these methods for the study of hematoxylin/eosin or immunohistochemically stained sections were not successful since the staining inhibited the PCR reactions. Only 25 of 50 samples were successfully amplified and of those only 14 produced accurate genotypes. Accurate genetic testing for polymorphisms in several genes of pharmacogenetic importance can be obtained from archival paraffin-embedded tumor samples. Thus, pharmacogenetic analysis can be applied to existing cancer therapy trials to test associations between these polymorphisms and treatment response

Does Unicondylar Knee Arthroplasty Affect Tibial Bone Strain? A Paired Cadaveric Comparison of Fixed- and Mobile-bearing Designs

BACKGROUND: Unexplained pain in the medial proximal tibia frequently leads to revision after unicondylar knee arthroplasty (UKA). As one of the most important factors for osteogenic adaptive response, increased bone strain following UKA has been suggested as a possible cause. QUESTIONS/PURPOSES: In this study we: (1) performed a cadaver-based kinematic analysis on paired cadaveric specimens before and after mobile-bearing and fixed-bearing UKA; and (2) simultaneously characterized the strain distribution in the anterior and posterior proximal tibia during squatting. METHODS: Five pairs of fresh, frozen full-leg cadaver specimens (four male, one female, 64 years to 87 years) were subjected to a dynamic squatting motion on a kinematic rig to simulate joint loading for a large ROM. Forces were applied to the quadriceps and hamstrings during the simulation while an infrared camera system tracked the location of reflective markers attached to the tibia and femur. Tibial cortical bone strain was measured with stacked strain gauge rosettes attached at predefined anterior and posterior positions on the medial cortex. Pairwise implantation of mobile-bearing (UKAMB) and fixed-bearing implants (UKAFB) allowed a direct comparison of right and left knees from the same donor through a linear mixed model. RESULTS: UKAMB more closely replicated native kinematics in terms of tibial rotation and in AP and mediolateral translation. Maximum principal bone strain values were consistently increased compared with native (anteromedial, mean [± SD] peak strain: 311 µε ± 190 and posterior, mean peak strain: 321 µε ± 147) with both designs in the anteromedial (UKAFB, mean peak strain: 551 µε ± 381, Cohen's d effect size 1.3 and UKAMB, mean peak strain: 596 µε ± 564, Cohen's d effect size 1.5) and posterior (UKAFB, mean peak strain: 505 µε ± 511, Cohen's d effect size 1.3 and UKAMB, mean peak strain: 633 µε ± 424, Cohen's d effect size 2.1) region. However, in the anterolateral region of the medial tibial bone, UKAFB demonstrated the overall largest increase in strain (mean peak strain: 1010 µε ± 787, Cohen's d effect size 1.9), while UKAMB (613 µε ± 395, Cohen's d effect size 0.2) closely replicated values of the native knee (563 µε ± 234). CONCLUSION: In this in vitro cadaver study both UKAMB and UKAFB led to an increase in bone strain in comparison with the native knee. However, in the anterolateral region of the medial tibial plateau, proximal tibial bone strain was lower after UKAMB and UKAFB. Both UKAMB and UKAFB lead to comparable increases in anteromedial and posterior tibial strain in comparison with the native knee. In the anterolateral region of the medial tibial plateau UKA, proximal tibial bone strain was closer to the native knee after UKAMB than after UKAFB. In an attempt to link kinematics and strain behavior of these designs there seemed to be no obvious relation. CLINICAL RELEVANCE: Further clinical research may be able to discern whether the observed differences in cortical strain after UKA is associated with unexplained pain in patients and whether the observed differences in cortical bone strain between mobile-bearing and fixed unicondylar designs results in a further difference in unexplained pain.status: publishe

4E-BP2/SH2B1/IRS2 Are Part of a Novel Feedback Loop That Controls β-Cell Mass

The mammalian target of rapamycin complex 1 (mTORC1) regulates several biological processes, although the key downstream mechanisms responsible for these effects are poorly defined. Using mice with deletion of eukaryotic translation initiation factor 4E-binding protein 2 (4E-BP2), we determine that this downstream target is a major regulator of glucose homeostasis and β-cell mass, proliferation, and survival by increasing insulin receptor substrate 2 (IRS2) levels and identify a novel feedback mechanism by which mTORC1 signaling increases IRS2 levels. In this feedback loop, we show that 4E-BP2 deletion induces translation of the adaptor protein SH2B1 and promotes the formation of a complex with IRS2 and Janus kinase 2, preventing IRS2 ubiquitination. The changes in IRS2 levels result in increases in cell cycle progression, cell survival, and β-cell mass by increasing Akt signaling and reducing p27 levels. Importantly, 4E-BP2 deletion confers resistance to cytokine treatment in vitro. Our data identify SH2B1 as a major regulator of IRS2 stability, demonstrate a novel feedback mechanism linking mTORC1 signaling with IRS2, and identify 4E-BP2 as a major regulator of proliferation and survival of β-cells

Novel roles of mTORC2 in regulation of insulin secretion by actin filament remodeling

Mammalian target of rapamycin (mTOR) kinase is an essential hub where nutrients and growth factors converge to control cellular metabolism. mTOR interacts with different accessory proteins to form complexes 1 and 2 (mTORC), and each complex has different intracellular targets. Although mTORC1's role in β-cells has been extensively studied, less is known about mTORC2's function in β-cells. Here, we show that mice with constitutive and inducible β-cell-specific deletion of RICTOR (; βRicKO; and i; βRicKO; mice, respectively) are glucose intolerant due to impaired insulin secretion when glucose is injected intraperitoneally. Decreased insulin secretion in βRicKO islets was caused by abnormal actin polymerization. Interestingly, when glucose was administered orally, no difference in glucose homeostasis and insulin secretion were observed, suggesting that incretins are counteracting the mTORC2 deficiency. Mechanistically, glucagon-like peptide-1 (GLP-1), but not gastric inhibitory polypeptide (GIP), rescued insulin secretion in vivo and in vitro by improving actin polymerization in; βRicKO; islets. In conclusion, mTORC2 regulates glucose-stimulated insulin secretion by promoting actin filament remodeling.; NEW & NOTEWORTHY; The current studies uncover a novel mechanism linking mTORC2 signaling to glucose-stimulated insulin secretion by modulation of the actin filaments. This work also underscores the important role of GLP-1 in rescuing defects in insulin secretion by modulating actin polymerization and suggests that this effect is independent of mTORC2 signaling