Faculty Bibliography

Acyl-CoA:diacylglycerol acyltransferase-1 (DGAT1) catalyzes the final step of triglyceride synthesis in mammalian cells. Data obtained from DGAT1-knockout mice have indicated that this enzyme plays an important role in energy homeostasis. We investigated the regulation of the expression and function of DGAT1 in mouse 3T3-L1 cell as a model for mammalian adipocytes. We demonstrated that the DGAT1 protein level increased by approximately 90-fold following differentiation of 3T3-L1 into mature adipocytes, a change that was accompanied by approximately 7-fold increase in DGAT1 mRNA. On the other hand, forced overexpression of DGAT1 mRNA by >20-fold via a recombinant adenovirus only resulted in approximately 2-fold increase in DGAT1 protein in mature adipocytes and little increase in preadipocytes. These results indicated that gene expression of DGAT1 in adipocytes is subjected to rigorous posttranscriptional regulation, which is modulated significantly by the differentiation status of 3T3-L1 cells. Protein stability is not a significant factor in the control of DGAT1 expression. The steady-state levels of DGAT1 were unaffected by blockage of proteolytic pathways by ALLN. However, translational control was suggested by sequence analysis of the 5'-untranslated region of human DGAT1 (hDGAT1) mRNA. We found that the level of DGAT1 activity was predominantly a function of the steady-state level of DGAT1 protein. No significant functional changes were observed when the conserved tyrosine phosphorylation site in hDGAT1 was mutated by a single base pair substitution. Despite only a approximately 2-fold increase in DGAT1 protein caused by recombinant viral transduction, a proportionate increase in cellular triglyceride synthesis resulted without affecting the triglyceride lipolysis rate, leading to >2-fold increase in intracellular triglyceride accumulation. No change in adipocyte morphology or in the expression levels of lipoprotein lipase, proxisomal proliferation-activating receptor-gamma, and aP2 was evident secondary to DGAT1 overexpression at different stages in 3T3-L1 differentiation. These data suggest that dysregulation of DGAT1 may play a role in the development of obesity, and manipulation of the steady-state level of DGAT1 protein may offer a potential means to treat or prevent obesity.

The regulation of signaling pathways by feedback inhibitors has become an emerging theme in the control of pattern formation during development. Nodal and Lefty proteins belong to divergent subfamilies of the TGF-beta family. Nodal signals promote mesendoderm induction in vertebrates, and Lefty proteins antagonize it. In zebrafish, Squint functions as a long-range Nodal signal during mesoderm induction. We report that the range over which Squint induces mesoderm is reduced by Lefty proteins. In contrast, the activity range of the short-range Nodal signal Cyclops is not regulated by Lefty activity. We present three lines of evidence that Lefty proteins diminish the range of Squint signaling by acting not only as antagonists of Squint autoregulation but also as long-range inhibitors of Squint activity. First, Lefty can block Nodal signaling at a distance. Second, Lefty regulates the range of Squint signaling before regulating squint expression. Third, Lefty restricts the range of Squint activity in squint mutant embryos, in which the endogenous gene is not subject to autoregulation. We also find that Lefty restricts the response to both high and low levels of Nodal signaling. These results indicate that Lefty proteins restrict the activity range of Nodal signals by dampening Nodal signaling in surrounding cells

Modification of low density lipoprotein (LDL) can result in the avid uptake of these lipoproteins via a family of macrophage transmembrane proteins referred to as scavenger receptors (SRs). The genetic inactivation of either of two SR family members, SR-A or CD36, has been shown previously to reduce oxidized LDL uptake in vitro and atherosclerotic lesions in mice. Several other SRs are reported to bind modified LDL, but their contribution to macrophage lipid accumulation is uncertain. We generated mice lacking both SR-A and CD36 to determine their combined impact on macrophage lipid uptake and to assess the contribution of other SRs to this process. We show that SR-A and CD36 account for 75-90% of degradation of LDL modified by acetylation or oxidation. Cholesteryl ester derived from modified lipoproteins fails to accumulate in macrophages taken from the double null mice, as assessed by histochemistry and gas chromatography-mass spectrometry. These results demonstrate that SR-A and CD36 are responsible for the preponderance of modified LDL uptake in macrophages and that other scavenger receptors do not compensate for their absence

The maturation of synaptic structures depends on inductive interactions between axons and their prospective targets. One example of such an interaction is the influence of proprioceptive sensory axons on the differentiation of muscle spindles. We have monitored the expression of three transcription factors, Egr3, Pea3, and Erm, that delineate early muscle spindle development in an assay of muscle spindle-inducing signals. We provide genetic evidence that Neuregulin1 (Nrg1) is required for proprioceptive afferent-evoked induction of muscle spindle differentiation in the mouse. Ig-Nrg1 isoforms are preferentially expressed by proprioceptive sensory neurons and are sufficient to induce muscle spindle differentiation in vivo, whereas CRD-Nrg1 isoforms are broadly expressed in sensory and motor neurons but are not required for muscle spindle induction

We present a new system for simultaneous morphological and molecular analysis of thick tissue samples. The system is composed of a computer-assisted microscope and a JAVA-based image display, analysis, and visualization program that allows acquisition, annotation, meaningful storage, three-dimensional reconstruction, and analysis of structures of interest in thick sectioned tissue specimens. We describe the system in detail and illustrate its use by imaging, reconstructing, and analyzing two complete tissue blocks that were differently processed and stained. One block was obtained from a ductal carcinoma in situ (DCIS) lumpectomy specimen and stained alternatively with Hematoxilyn and Eosin (H&E), and with a counterstain and fluorescence in situ hybridization (FISH) to the ERB-B2 gene. The second block contained a fully sectioned mammary gland of a mouse, stained for histology with H&E. We show how the system greatly reduces the amount of interaction required for the acquisition and analysis and is, therefore, suitable for studies that require morphologically driven, wide-scale (e.g., whole gland) analysis of complex tissue samples or cultures

Anatomy remains one of the core courses of medical school, but the time devoted to it is decreasing. To accommodate the explosion of medical knowledge, educators search to streamline the curriculum. Because it is time-consuming, dissection comes under increased scrutiny. Even in the face of these pressures to reduce course hours, I would like to propose broadening, not reducing, the responsibilities of the anatomy instructor. Anatomy instructors can play a crucial role in helping medical schools meet the critical need to cultivate humanistic values, especially in the arena of end-of-life care. Anatomy can--and should--play an important role in a curriculum-wide effort to address this issue. Just as dissection remains an essential technique to teach three-dimensional concepts, the cadaver dissection lab is an ideal place to introduce concepts of humanistic care. The lab evokes the students' memories, speculations, and fears about serious illness in themselves, their families, and loved ones. Some programs address these reactions with supplemental activities, such as journaling, essay writing, and small group discussion. Valuable as these activities may be, anatomy instructors can achieve more by recognizing their role as a mentor, who can integrate humanistic values into traditional course objectives in a way that adds little time to the curriculum. The attitude of the instructor in ministering to the students' needs as they undertake the emotionally charged task of dissection can provide a model for how the students will respond, in turn, to the hopes and fears of their patients-and to their own reactions to dying. This approach will allow students to implement and practice humanistic values immediately, laying a foundation for their clinical training.

6-hydroxydopamine, 1-methyl-4-phenyl-pyridinium (MPP+), and rotenone cause the death of dopaminergic neurons in vitro and in vivo and are widely used to model Parkinson's disease. To identify regulated genes in such models, we performed serial analysis of gene expression on neuronal PC12 cells exposed to 6-hydroxydopamine. This revealed a striking increase in transcripts associated with the unfolded protein response. Immunoblotting confirmed phosphorylation of the key endoplasmic reticulum stress kinases IRE1alpha and PERK (PKR-like ER kinase) and induction of their downstream targets. There was a similar response to MPP+ and rotenone, but not to other apoptotic initiators. As evidence that endoplasmic reticulum stress contributes to neuronal death, sympathetic neurons from PERK null mice in which the capacity to respond to endoplasmic reticulum stress is compromised were more sensitive to 6-hydroxydopamine. Our findings, coupled with evidence from familial forms of Parkinson's disease, raise the possibility of widespread involvement of endoplasmic reticulum stress and the unfolded protein response in the pathophysiology of this disease.

BACKGROUND:The cytoskeleton and associated motors play an important role in the establishment of intracellular polarity. Microtubule-based transport is required in many cell types for the asymmetric localization of mRNAs and organelles. A striking example is the Drosophila oocyte, where microtubule-dependent processes govern the asymmetric positioning of the nucleus and the localization to distinct cortical domains of mRNAs that function as cytoplasmic determinants. A conserved machinery for mRNA localization and nuclear positioning involving cytoplasmic Dynein has been postulated; however, the precise role of plus- and minus end-directed microtubule-based transport in axis formation is not yet understood. RESULTS:Here, we show that mRNA localization and nuclear positioning at mid-oogenesis depend on two motor proteins, cytoplasmic Dynein and Kinesin I. Both of these microtubule motors cooperate in the polar transport of bicoid and gurken mRNAs to their respective cortical domains. In contrast, Kinesin I-mediated transport of oskar to the posterior pole appears to be independent of Dynein. Beside their roles in RNA transport, both motors are involved in nuclear positioning and in exocytosis of Gurken protein. Dynein-Dynactin complexes accumulate at two sites within the oocyte: around the nucleus in a microtubule-independent manner and at the posterior pole through Kinesin-mediated transport. CONCLUSION/CONCLUSIONS:The microtubule motors cytoplasmic Dynein and Kinesin I, by driving transport to opposing microtubule ends, function in concert to establish intracellular polarity within the Drosophila oocyte. Furthermore, Kinesin-dependent localization of Dynein suggests that both motors are components of the same complex and therefore might cooperate in recycling each other to the opposite microtubule pole.

P58(IPK) is an Hsp40 family member known to inhibit the interferon (IFN)-induced, double-stranded RNA-activated, eukaryotic initiation factor 2alpha (eIF2alpha) protein kinase R (PKR) by binding to its kinase domain. We find that the stress of unfolded proteins in the endoplasmic reticulum (ER) activates P58(IPK) gene transcription through an ER stress-response element in its promoter region. P58(IPK) interacts with and inhibits the PKR-like ER-localized eIF2alpha kinase PERK, which is normally activated during the ER-stress response to protect cells from ER stress by attenuating protein synthesis and reducing ER client protein load. Levels of phosphorylated eIF2alpha were lower in ER-stressed P58(IPK)-overexpressing cells and were enhanced in P58(IPK) mutant cells. In the ER-stress response, PKR-like ER kinase (PERK)-mediated translational repression is transient and is followed by translational recovery and enhanced expression of genes that increase the capacity of the ER to process client proteins. The absence of P58(IPK) resulted in increased expression levels of two ER stress-inducible genes, BiP and Chop, consistent with the enhanced eIF2alpha phosphorylation in the P58(IPK) deletion cells. Our studies suggest that P58(IPK) induction during the ER-stress response represses PERK activity and plays a functional role in the expression of downstream markers of PERK activity in the later phase of the ER-stress response.

beta-Amyloid accumulation is associated with pathologic changes in the brain in Alzheimer's disease and has recently been identified in plaques of another chronic inflammatory disorder, atherosclerosis. The class B scavenger receptor, CD36, mediates binding of fibrillar beta-amyloid to cells of the monocyte/macrophage lineage, including brain macrophages (microglia). In this study, we demonstrate that in microglia and other tissue macrophages, beta-amyloid initiates a CD36-dependent signaling cascade involving the Src kinase family members, Lyn and Fyn, and the mitogen-activated protein kinase, p44/42. Interruption of this signaling cascade, through targeted disruption of Src kinases downstream of CD36, inhibits macrophage inflammatory responses to beta-amyloid, including reactive oxygen and chemokine production, and results in decreased recruitment of microglia to sites of amyloid deposition in vivo. The finding that engagement of CD36 by beta-amyloid initiates a Src kinase-dependent production of inflammatory mediators in cells of the macrophage lineage reveals a novel receptor-mediated pro-inflammatory signaling pathway of potential therapeutic importance