Faculty Bibliography

Ischemia-induced acidification of astrocytes or cardiac myocytes reduces intercellular communication by closing gap junction channels and subsequently internalizing gap junction proteins. To determine whether such coupling changes might be attributable to altered interactions between connexin43 (Cx43) and other proteins, we applied the nigericin/high K+ method to vary intracellular pH (pHi) in cultured cortical astrocytes. Intracellular acidification was accompanied by internalization of Cx43 with retention of Cx43 scaffolding protein Zonula Occludens-1 (ZO-1) at cell surfaces, suggesting that ZO-1 and Cx43 dissociate at low pHi. Coimmunoprecipitation studies revealed decreased binding of ZO-1 and increased binding of c-Src to Cx43 at low pHi. Resonant mirror spectroscopy was used to quantify binding of the SH3 domain of c-Src and the PDZ domains of ZO-1 to the carboxyl terminal domain of Cx43 (Cx43CT). Data indicate that the c-Src/Cx43CT interaction is highly pH dependent whereas the ZO-1/Cx43CT interaction is not. Moreover, binding of c-Src to Cx43CT prevented and reversed ZO-1/Cx43CT binding. We hypothesize that increased affinity of c-Src for Cx43 at low pHi aids in separation of Cx43 from ZO-1 and that this may facilitate internalization of Cx43. These data suggest that protracted acidification may remodel protein-protein interactions involving Cx43 and thus provide an important protective mechanism to limit lesion spread after ischemic injury

Accumulating evidence has indicated that neurotrophin receptor trafficking plays an important role in neurotrophin-mediated signaling in developing as well as mature neurons. However, little is known about the molecular mechanisms and the components of neurotrophin receptor vesicular transport. This article will describe how neurotrophin receptors, Trk and p75 neurotrophin receptor (p75NTR), are intimately involved in the axonal transport process. In particular, the molecules that may direct Trk receptor trafficking in the axon will be discussed. Finally, potential mechanisms by which receptor-containing vesicles link to molecular cytoskeletal motors will be presented

Rap1 and Ras are closely related GTPases that share some effectors but have distinct functions. We studied the subcellular localization of Rap1 and its sites of activation in living cells. Both GFP-tagged Rap1 and endogenous Rap1 were localized to the plasma membrane (PM) and endosomes. The PM association of GFP-Rap1 was dependent on GTP binding, and GFP-Rap1 was rapidly up-regulated on this compartment in response to mitogens, a process blocked by inhibitors of endosome recycling. A novel fluorescent probe for GTP-bound Rap1 revealed that this GTPase was transiently activated only on the PM of both fibroblasts and T cells. Activation on the PM was blocked by inhibitors of endosome recycling. Moreover, inhibition of endosome recycling blocked the ability of Rap1 to promote integrin-mediated adhesion of T cells. Thus, unlike Ras, the membrane localizations of Rap1 are dynamically regulated, and the PM is the principle platform from which Rap1 signaling emanates. These observations may explain some of the biological differences between these GTPases

BACKGROUND: Stress triggers adaptive and maladaptive changes in the central nervous system, including activation of the hypothalamic-pituitary-adrenal axis, and can trigger mood disorders and posttraumatic stress disorder. We examined the effect of immobilization stress (IMO) on gene expression of tryptophan hydroxylase (TPH), the rate-limiting enzyme in serotonin biosynthesis, and the role of cortisol in that response. METHODS: Regular and adrenalectomized Sprague-Dawley rats were exposed to various repetitions of IMO. Tryptophan hydroxylase messenger ribonucleic acid (mRNA) was determined by competitive reverse transcriptase polymerase chain reaction, and TPH protein was examined by immunoblot and immunocytochemistry. RESULTS: Elevation of TPH mRNA by IMO was tissue-specific and dose-dependent. A single IMO elicited a threefold rise in TPH mRNA in median raphe nucleus (MRN), but repeated (3x) IMOs were needed for similar response in dorsal raphe nucleus (DRN). Repeated daily IMO, up to 7 days, triggered a robust induction (6-10-fold) in TPH mRNA, accompanied by corresponding rise in TPH protein levels in raphe nuclei but not in the pineal gland. The rise in TPH immunoreactivity was widespread throughout the DRN and MRN. Bilateral adrenalectomy did not prevent the IMO-triggered increase in TPH immunoreactive protein in the raphe nuclei. CONCLUSIONS: This study reveals adrenal glucocorticoid-independent induction of TPH gene expression in raphe nuclei in response to immobilization stress

Mutations in the human P gene result in oculocutaneous albinism type 2, the most common form of albinism. Mouse melan-p1 melanocytes, cultured from mice null at the homologous pink-eyed dilution (p) locus, exhibit defective melanin production. A variety of compounds including tyrosine, NH4Cl, bafilomycin A1, concanamycin, monensin, and nigericin are capable of restoring melanin synthesis in these cells. In the current study, we investigated the subcellular effects of bafilomycin A1 and monensin treatment of melan-p1 cells. Both agents play two roles in the processing of tyrosinase (Tyr) in melan-p1 cells. First, combined glycosidase digestion and immunoblotting analysis showed that these agents reduce levels of Tyr retained in the endoplasmic reticulum (ER) and facilitate the release of Tyr from the ER to the Golgi. Secondly, treatment with these compounds resulted in the stabilization of Tyr. Surprisingly, induction of melanin synthesis corresponds more closely with diminution of ER-retained Tyr, rather than the absolute amount of Tyr. Our results suggest that bafilomycin A1 and monensin induce melanin synthesis in melan-p1 cells mainly by facilitating Tyr processing from the ER to the Golgi by increasing the pH in either the ER or the ER-Golgi intermediate compartment

In response to environmental stress, cells induce a program of gene expression designed to remedy cellular damage or, alternatively, induce apoptosis. In this report, we explore the role of a family of protein kinases that phosphorylate eukaryotic initiation factor 2 (eIF2) in coordinating stress gene responses. We find that expression of activating transcription factor 3 (ATF3), a member of the ATF/CREB subfamily of basic-region leucine zipper (bZIP) proteins, is induced in response to endoplasmic reticulum (ER) stress or amino acid starvation by a mechanism requiring eIF2 kinases PEK (Perk or EIF2AK3) and GCN2 (EIF2AK4), respectively. Increased expression of ATF3 protein occurs early in response to stress by a mechanism requiring the related bZIP transcriptional regulator ATF4. ATF3 contributes to induction of the CHOP transcriptional factor in response to amino acid starvation, and loss of ATF3 function significantly lowers stress-induced expression of GADD34, an eIF2 protein phosphatase regulatory subunit implicated in feedback control of the eIF2 kinase stress response. Overexpression of ATF3 in mouse embryo fibroblasts partially bypasses the requirement for PEK for induction of GADD34 in response to ER stress, further supporting the idea that ATF3 functions directly or indirectly as a transcriptional activator of genes targeted by the eIF2 kinase stress pathway. These results indicate that ATF3 has an integral role in the coordinate gene expression induced by eIF2 kinases. Given that ATF3 is induced by a very large number of environmental insults, this study supports involvement of eIF2 kinases in the coordination of gene expression in response to a more diverse set of stress conditions than previously proposed

Beta-catenin and cyclin D1 have attracted considerable attention due to their proto-oncogenic roles in human cancer. The finding of cyclin D1 as a direct target gene of beta-catenin in colon cancer cells led to the assumption that cyclin D1 upregulation is pivotal to beta-catenin's oncogenicity. Our recent paper shows that this is not the case; cyclin D1 dampens the oncogenicity of activated beta-catenin (MMTV-DN89beta-catenin). The relationships and dependencies of beta-catenin and cyclin D1 point to distinct, essential and sequential roles during alveologenesis. These results support the concept that both beta-catenin's and cyclin D1's actions are more sophisticated than simple acceleration of the cell cycle clock. These proteins are employed at critical junctures involving cell fate decisions that we speculate require specific types of cell cycle to traverse

In menopausal women and the elderly, populations most often affected by osteoarthritis (OA), estrogen levels are lower than normal, which suggests that estrogen may be an important regulator of OA. Estrogen can influence chondrocyte function on multiple levels by interacting with cellular growth factors, adhesion molecules, and cytokines. Nevertheless, findings regarding a correlation between estrogen and OA are inconsistent and inconclusive and range from estrogen protecting against OA to cartilage damage mediated by high levels of estrogen and higher binding to estrogen receptors. In this review, we summarize current in vivo and in vitro research and discuss future directions for analyses of the role of estrogen in OA

Successful cell migration depends on the careful regulation of the timing of movement, the guidance of motile cells, and cytoskeletal and adhesive changes within the cells. This review focuses on genes that act cell-autonomously to promote these aspects of cell migration in Drosophila. We discuss recent advances in understanding the migration of the ovarian border cells, embryonic blood cells, primordial germ cells, somatic gonadal precursors, and tracheal cells. Comparison of genes that regulate these processes to those that promote tumorigenesis and metastasis in mammals demonstrates that studies in fruit flies are uncovering new genes highly relevant to cancer biology.