Faculty Bibliography

STEAP1, six-transmembrane epithelial antigen of prostate member 1, is strongly expressed in several types of cancer cells, particularly in prostate cancer, and inhibition of its expression reduces the rate of tumor cell proliferation. However, the physiological function of STEAP1 remains unknown. Here for the first time, we purified a mammalian (rabbit) STEAP1 at a milligram level, permitting its high-quality biochemical and biophysical characterizations. We found that STEAP1 likely assembles as a homotrimer and forms a heterotrimer when co-expressed with STEAP2. Each STEAP1 protomer binds one heme prosthetic group that is mainly low-spin with a pair of histidine axial ligands, with small portions of high-spin and P450-type heme. In its ferrous state, STEAP1 is capable of reducing transition metal ion complexes of Fe3+ and Cu2+. Ferrous STEAP1 also reacts readily with O2 through an outer sphere redox mechanism. Kinetics with all three substrates are biphasic with approximately 80 and approximately 20% for the fast and slow phases, respectively, in line with its heme heterogeneity. STEAP1 retained a low level of bound FAD during purification, and the binding equilibrium constant, KD, was approximately 30 muM. These results highlight STEAP as a novel metal reductase and superoxide synthase and establish a solid basis for further research into understanding how STEAP1 activities may affect cancer progression.

Maintenance of mammary functional capacity during cycles of proliferation and regression depends on appropriate cell fate decisions of mammary progenitor cells to populate an epithelium consisting of secretory luminal cells and contractile myoepithelial cells. It is well established that transforming growth factor-beta (TGFbeta) restricts mammary epithelial cell proliferation and that sensitivity to TGFbeta is decreased in breast cancer. We show that TGFbeta also exerts control of mammary progenitor self-renewal and lineage commitment decisions by stringent regulation of breast cancer associated 1 (BRCA1), which controls stem cell self-renewal and lineage commitment. Either genetic depletion of Tgfb1 or transient blockade of TGFbeta increased self-renewal of mammary progenitor cells in mice, cultured primary mammary epithelial cells, and also skewed lineage commitment toward the myoepithelial fate. TGFbeta stabilized the abundance of BRCA1 by reducing the abundance of microRNA-182 (miR-182). Ectopic expression of BRCA1 or antagonism of miR-182 in cultured TGFbeta-deficient mammary epithelial cells restored luminal lineage commitment. These findings reveal that TGFbeta modulation of BRCA1 directs mammary epithelial cell fate and, because stem or progenitor cells are thought to be the cell of origin for aggressive breast cancer subtypes, suggest that TGFbeta dysregulation during tumorigenesis may promote distinct breast cancer subtypes.

Inherited mtDNA mutations cause severe human disease. In most species, mitochondria are inherited maternally through mechanisms that are poorly understood. Genes that specifically control the inheritance of mitochondria in the germline are unknown. Here, we show that the long isoform of the protein Oskar regulates the maternal inheritance of mitochondria in Drosophila melanogaster. We show that, during oogenesis, mitochondria accumulate at the oocyte posterior, concurrent with the bulk streaming and churning of the oocyte cytoplasm. Long Oskar traps and maintains mitochondria at the posterior at the site of primordial germ cell (PGC) formation through an actin-dependent mechanism. Mutating long oskar strongly reduces the number of mtDNA molecules inherited by PGCs. Therefore, Long Oskar ensures germline transmission of mitochondria to the next generation. These results provide molecular insight into how mitochondria are passed from mother to offspring, as well as how they are positioned and asymmetrically partitioned within polarized cells.

The roles of prolyl hydroxylase domain proteins (PHDs) in bone are incompletely understood. Here we deleted the expression of genes encoding PHD1, PHD2, and PHD3 in osteoblasts in mice by breeding the floxed Phd1-3 mice with Col1a1-Cre transgenic mice. Results showed that mice lacking PHD1-3 in osteoblasts (Phd1-3ob-/-) had increased bone mass. Bone parameters such as bone volume/tissue volume (BV/TV), trabecular number (Tb.N), and trabecular thickness (Tb.Th) were increased, while trabecular spacing (Tb.Sp) was decreased in Phd1-3ob-/- relative to wild-type (WT) femurs. In contrast, loss of PHD1-3 in osteoblasts did not alter cortical thickness (Cort.Th). The mineralization apposition rate (MAR) was increased in Phd1-3ob-/- bone compared to that of wild-type (WT) bone, demonstrating an enhancement of osteoblast function. Loss of PHD1-3 increased the number of osteoblast progenitors (CFU-OBs) in bone marrow cultures. Interestingly, deleting Phd1-3 genes in osteoblasts increased osteoclast formation in vitro and in bone.

Rapid impulse propagation in the heart is a defining property of pectinated atrial myocardium (PAM) and the ventricular conduction system (VCS) and is essential for maintaining normal cardiac rhythm and optimal cardiac output. Conduction defects in these tissues produce a disproportionate burden of arrhythmic disease and are major predictors of mortality in heart failure patients. Despite the clinical importance, little is known about the gene regulatory network that dictates the fast conduction phenotype. Here, we have used signal transduction and transcriptional profiling screens to identify a genetic pathway that converges on the NRG1-responsive transcription factor ETV1 as a critical regulator of fast conduction physiology for PAM and VCS cardiomyocytes. Etv1 was highly expressed in murine PAM and VCS cardiomyocytes, where it regulates expression of Nkx2-5, Gja5, and Scn5a, key cardiac genes required for rapid conduction. Mice deficient in Etv1 exhibited marked cardiac conduction defects coupled with developmental abnormalities of the VCS. Loss of Etv1 resulted in a complete disruption of the normal sodium current heterogeneity that exists between atrial, VCS, and ventricular myocytes. Lastly, a phenome-wide association study identified a link between ETV1 and bundle branch block and heart block in humans. Together, these results identify ETV1 as a critical factor in determining fast conduction physiology in the heart.

Hematopoietic-specific transcription factors require coactivators to communicate with the general transcription machinery and establish transcriptional programs that maintain hematopoietic stem cell (HSC) self-renewal, promote differentiation, and prevent malignant transformation. Mediator is a large coactivator complex that bridges enhancer-localized transcription factors with promoters, but little is known about Mediator function in adult stem cell self-renewal and differentiation. We show that MED12, a member of the Mediator kinase module, is an essential regulator of HSC homeostasis, as in vivo deletion of Med12 causes rapid bone marrow aplasia leading to acute lethality. Deleting other members of the Mediator kinase module does not affect HSC function, suggesting kinase-independent roles of MED12. MED12 deletion destabilizes P300 binding at lineage-specific enhancers, resulting in H3K27Ac depletion, enhancer de-activation, and consequent loss of HSC stemness signatures. As MED12 mutations have been described recently in blood malignancies, alterations in MED12-dependent enhancer regulation may control both physiological and malignant hematopoiesis.

The development of the mammalian cerebellum is orchestrated by both cell-autonomous programs and inductive environmental influences. Here, we describe the main processes of cerebellar ontogenesis, highlighting the neurogenic strategies used by developing progenitors, the genetic programs involved in cell fate specification, the progressive changes of structural organization, and some of the better-known abnormalities associated with developmental disorders of the cerebellum.

PURPOSE OF REVIEW:Better tools are sorely needed for both the prevention and treatment of cardiovascular diseases, which account for more than one-third of the deaths in Western countries. MicroRNAs typically regulate the expression of several mRNAs involved in the same biological process. Therapeutic manipulation of miRNAs could restore the expression of multiple players within the same physiologic pathway, and ideally offer better curative outcomes than conventional approaches that target only one single player within the pathway. This review summarizes available studies on the prospective value of targeting miRNAs to prevent dyslipidemia and atherogenesis. RECENT FINDINGS:Silencing the expression of miRNAs that target key genes involved in lipoprotein metabolism in vivo with antisense oligonucleotides results in the expected de-repression of target mRNAs in liver and atherosclerotic plaques. However, the consequences of long-term antimiRNA treatment on both circulating lipoproteins and athero-protection are yet to be established. SUMMARY:A number of studies have demonstrated the efficacy of miRNA mimics and inhibitors as novel therapeutic tools for treating dyslipidemia and cardiovascular diseases. Nevertheless, concerns over unanticipated side-effects related to de-repression of additional targets should not be overlooked for miRNA-based therapies.

AIMS: To evaluate whether an ultra-low-dose CT protocol can diagnose selected limb fractures as well as conventional CT (C-CT). PATIENTS AND METHODS: We prospectively studied 40 consecutive patients with a limb fracture in whom a CT scan was indicated. These were scanned using an ultra-low-dose CT Reduced Effective Dose Using Computed Tomography In Orthopaedic Injury (REDUCTION) protocol. Studies from 16 selected cases were compared with 16 C-CT scans matched for age, gender and type of fracture. Studies were assessed for diagnosis and image quality. Descriptive and reliability statistics were calculated. The total effective radiation dose for each scanned site was compared. RESULTS: The mean estimated effective dose (ED) for the REDUCTION protocol was 0.03 milliSieverts (mSv) and 0.43 mSv (p < 0.005) for C-CT. The sensitivity (Sn), specificity (Sp), positive predictive value (PPV) and negative predictive value (NPV) of the REDUCTION protocol to detect fractures were 0.98, 0.89, 0.98 and 0.89 respectively when two occult fractures were excluded. Inter- and intra-observer reliability for diagnosis using the REDUCTION protocol (kappa = 0.75, kappa = 0.71) were similar to those of C-CT (kappa = 0.85, kappa = 0.82). Using the REDUCTION protocol, 3D CT reconstructions were equivalent in quality and diagnostic information to those generated by C-CT (kappa = 0.87, kappa = 0.94). CONCLUSION: With a near 14-fold reduction in estimated ED compared with C-CT, the REDUCTION protocol reduces the amount of CT radiation substantially without significant diagnostic decay. It produces images that appear to be comparable with those of C-CT for evaluating fractures of the limbs. Cite this article: Bone Joint J 2016;98-B:1668-73.