Faculty Bibliography

To identify molecular mechanisms that function in G-protein signaling, we have performed molecular genetic studies of a simple behavior of the nematode Caenorhabditis elegans, egg laying, which is driven by a pair of serotonergic neurons, the hermaphrodite-specific neurons (HSNs). The activity of the HSNs is regulated by the G(o)-coupled receptor EGL-6, which mediates inhibition of the HSNs by neuropeptides. We report here that this inhibition requires one of three inwardly rectifying K(+) channels encoded by the C. elegans genome: IRK-1. Using ChannelRhodopsin-2-mediated stimulation of HSNs, we observed roles for egl-6 and irk-1 in regulating the excitability of HSNs. Although irk-1 is required for inhibition of HSNs by EGL-6 signaling, we found that other G(o) signaling pathways that inhibit HSNs involve irk-1 little or not at all. These findings suggest that the neuropeptide receptor EGL-6 regulates the potassium channel IRK-1 via a dedicated pool of G(o) not involved in other G(o)-mediated signaling. We conclude that G-protein-coupled receptors that signal through the same G-protein in the same cell might activate distinct effectors and that specific coupling of a G-protein-coupled receptor to its effectors can be determined by factors other than its associated G-proteins.

Staphylococcus aureus peptidoglycan (PG) is densely functionalized with anionic polymers called wall teichoic acids (WTAs). These polymers contain three tailoring modifications: d-alanylation, alpha-O-GlcNAcylation, and beta-O-GlcNAcylation. Here we describe the discovery and biochemical characterization of a unique glycosyltransferase, TarS, that attaches beta-O-GlcNAc (beta-O-N-acetyl-D-glucosamine) residues to S. aureus WTAs. We report that methicillin resistant S. aureus (MRSA) is sensitized to beta-lactams upon tarS deletion. Unlike strains completely lacking WTAs, which are also sensitive to beta-lactams, DeltatarS strains have no growth or cell division defects. Because neither alpha-O-GlcNAc nor beta-O-Glucose modifications can confer resistance, the resistance phenotype requires a highly specific chemical modification of the WTA backbone, beta-O-GlcNAc residues. These data suggest beta-O-GlcNAcylated WTAs scaffold factors required for MRSA resistance. The beta-O-GlcNAc transferase identified here, TarS, is a unique target for antimicrobials that sensitize MRSA to beta-lactams.

Blood vessels deliver oxygen, nutrients, hormones and immunity factors throughout the body. To perform these vital functions, vascular cords branch, lumenize and interconnect. Yet, little is known about the cellular, molecular and physiological mechanisms that control how circulatory networks form and interconnect. Specifically, how circulatory networks merge by interconnecting in parallel along their boundaries remains unexplored. To examine this process we studied the formation and functional maturation of the plexus that forms between the Dorsal Longitudinal Anastomotic Vessels (DLAVs) in the zebrafish. We find that the migration and proliferation of endothelial cells within the DLAVs and their Segmental (Se) vessel precursors drives DLAV plexus formation. Remarkably, the presence of Se vessels containing only endothelial cells of the arterial lineage is sufficient for DLAV plexus morphogenesis, suggesting that endothelial cells from the venous lineage make a dispensable or null contribution to this process. The discovery of a circuit that integrates the inputs of circulatory flow and Vascular Endothelial Growth Factor (VEGF) signaling to modulate aortic arch angiogenesis, together with the expression of components of this circuit in the trunk vasculature, prompted us to interrogate the role of these inputs and their relationship during DLAV plexus formation. We find that circulatory flow and VEGF signaling make additive contributions to DLAV plexus morphogenesis, rather than acting as essential inputs with equivalent contributions as they do during aortic arch angiogenesis. Our observations underscore the existence of context-dependent differences in the integration of physiological stimuli and signaling cascades during vascular development.

ML-IAP [melanoma IAP (inhibitor of apoptosis)] is an anti-apoptotic protein that is expressed highly in melanomas where it contributes to resistance to apoptotic stimuli. The anti-apoptotic activity and elevated expression of IAP family proteins in many human cancers makes IAP proteins attractive targets for inhibition by cancer therapeutics. Small-molecule IAP antagonists that bind with high affinities to select BIR (baculovirus IAP repeat) domains have been shown to stimulate auto-ubiquitination and rapid proteasomal degradation of c-IAP1 (cellular IAP1) and c-IAP2 (cellular IAP2). In the present paper, we report ML-IAP proteasomal degradation in response to bivalent, but not monovalent, IAP antagonists. This degradation required ML-IAP ubiquitin ligase activity and was independent of c-IAP1 or c-IAP2. Although ML-IAP is best characterized in melanoma cells, we show that ML-IAP expression in normal mammalian tissues is restricted largely to the eye, being most abundant in ciliary body epithelium and retinal pigment epithelium. Surprisingly, given this pattern of expression, gene-targeted mice lacking ML-IAP exhibited normal intraocular pressure as well as normal retinal structure and function. The results of the present study indicate that ML-IAP is dispensable for both normal mouse development and ocular homoeostasis.

Radiation therapy has the potential to convert the tumor into an in situ individualized vaccine by inducing immunogenic cancer cell death and pro-inflammatory cytokines and chemokines; however this potential is rarely realized by irradiation alone. We hypothesized that radiation-induced immunosuppressive factors may hinder its pro-immunogenic effects. Transforming growth factor beta (TGFbeta) has immunosuppressive function for dendritic cells and T cells and is activated by radiation. Here we tested the hypothesis that inhibiting TGFbeta during radiation treatment would induce an immunogenic response. Poorly immunogenic, highly metastatic 4T1 carcinoma cells were injected s.c. in syngeneic BALB/c mice (day 0). TGFbeta neutralizing 1D11 or isotype control 13C4 monoclonal antibodies were given i.p. (200 mg/mouse) every other day from day 12 to 28. Tumors were irradiated with 6Gy on five consecutive days beginning on day 13. Tumor growth was measured consecutively. Mice were euthanized at day 21 for analysis, at day 28 for enumeration of lung metastases, or followed for survival. Gene expression profiles were obtained using Affymetrix mouse genome 430 2.0 array. Tumor growth rates and the frequency of lung metastases were similar in mice receiving control antibody or 1D11 alone. Radiation treatment caused significant (P=0.0065) tumor growth delay but did not inhibit lung metastases. In contrast, mice treated with both 1D11 and radiation exhibited significantly greater tumor growth control and reduced lung metastases (P<0.0001), and significantly prolonged survival (P<0.005). As expected, TGFbeta signalling was inhibited with 1D11 as measured in CD4+ and CD8+ T cells from tumor-draining lymph nodes at day 21. CD8+ T cells producing IFN in response to a tumor-specific antigen were detected only in mice treated with 1D11 and radiation. Expression profiles showed that genes associated with immune response and T cell activation were upregulated in irradiated tumors of mice treated with 1D11 compared to other treatment groups. In vivo depletion experiments demonstrated that T cells were essential for the improved tumor control and inhibition of lung metastases of mice treated with 1D11 and radiation. These data support a critical role for TGFbeta as a regulator of the pro-immunogenic effects of local tumor radiotherapy. Inhibition of TGFbeta during radiotherapy may promote self-immunization and achieve systemic control of metastatic disease. Supported by DOD BCRP grant BC100481P2

Parathyroid hormone-related protein (PTHrP) regulates cell fate and specifies the mammary mesenchyme during embryonic development. Loss of PTHrP or its receptor (Pthr1) abolishes the expression of mammary mesenchyme markers and allows mammary bud cells to revert to an epidermal fate. By contrast, overexpression of PTHrP in basal keratinocytes induces inappropriate differentiation of the ventral epidermis into nipple-like skin and is accompanied by ectopic expression of Lef1, beta-catenin and other markers of the mammary mesenchyme. In this study, we document that PTHrP modulates Wnt/beta-catenin signaling in the mammary mesenchyme using a Wnt signaling reporter, TOPGAL-C. Reporter expression is completely abolished by loss of PTHrP signaling and ectopic reporter activity is induced by overexpression of PTHrP. We also demonstrate that loss of Lef1, a key component of the Wnt pathway, attenuates the PTHrP-induced abnormal differentiation of the ventral skin. To characterize further the contribution of canonical Wnt signaling to embryonic mammary development, we deleted beta-catenin specifically in the mammary mesenchyme. Loss of mesenchymal beta-catenin abolished expression of the TOPGAL-C reporter and resulted in mammary buds with reduced expression of mammary mesenchyme markers and impaired sexual dimorphism. It also prevented the ectopic, ventral expression of mammary mesenchyme markers caused by overexpression of PTHrP in basal keratinocytes. Therefore, we conclude that a mesenchymal, canonical Wnt pathway mediates the PTHrP-dependent specification of the mammary mesenchyme.

OBJECTIVES: The aims of this work were to study the epidemiological profiles, differences in echinocandin susceptibilities and clinical relevance of the Candida parapsilosis sensu lato species isolated from proven fungaemia cases at La Fe University Hospital of Valencia (Spain) from 1995 to 2007. RESULTS: The prevalence of these species was: C. parapsilosis sensu stricto, 74.4%; Candida orthopsilosis, 23.54%; and Candida metapsilosis, 2.05%. The incidence of the species complex as agents of fungaemia remained stationary until 2005 and doubled in 2006. The incidence of C. orthopsilosis showed an increasing trend during the study period, while C. parapsilosis sensu stricto incidence diminished. Also, an important epidemiological change was observed starting in 2004, when 86.5% of the C. parapsilosis sensu lato strains were found in adult patients, while before that year only 13.5% of the isolates were found in this population. CONCLUSIONS: Echinocandin drug susceptibility testing using the CLSI M27-A3 document showed a wide range of MIC values (0.015-4 mg/L), with micafungin being the most potent in vitro inhibitor followed by anidulafungin and caspofungin (MIC geometric mean of 0.68, 0.74 and 0.87 mg/L, respectively). C. metapsilosis was the most susceptible species of the complex to anidulafungin and micafungin in vitro (MIC(50) for anidulafungin and micafungin: 0.06 mg/L), while there were no differences between C. parapsilosis sensu lato species when caspofungin MIC(50)s were compared (MIC(50) 1.00 mg/L). Differences in caspofungin in vitro susceptibility were observed between the different clinical service departments of La Fe Hospital.

Environmental factors such as diets rich in saturated fats contribute to dysfunction and death of pancreatic beta-cells in diabetes. Endoplasmic reticulum (ER) stress is elicited in beta-cells by saturated fatty acids. Here we show that palmitate-induced beta-cell apoptosis is mediated by the intrinsic mitochondrial pathway. By microarray analysis, we identified a palmitate-triggered ER stress gene expression signature and the induction of the BH3-only proteins death protein 5 (DP5) and p53-upregulated modulator of apoptosis (PUMA). Knockdown of either protein reduced cytochrome c release, caspase-3 activation, and apoptosis in rat and human beta-cells. DP5 induction depends on inositol-requiring enzyme 1 (IRE1)-dependent c-Jun NH(2)-terminal kinase and PKR-like ER kinase (PERK)-induced activating transcription factor (ATF3) binding to its promoter. PUMA expression is also PERK/ATF3-dependent, through tribbles 3 (TRB3)-regulated AKT inhibition and FoxO3a activation. DP5(-/-) mice are protected from high fat diet-induced loss of glucose tolerance and have twofold greater pancreatic beta-cell mass. This study elucidates the crosstalk between lipotoxic ER stress and the mitochondrial pathway of apoptosis that causes beta-cell death in diabetes.

Pancreatic ductal adenocarcinoma is an aggressive cancer that interacts with stromal cells to produce a highly inflammatory tumor microenvironment that promotes tumor growth and invasiveness. The precise interplay between tumor and stroma remains poorly understood. TLRs mediate interactions between environmental stimuli and innate immunity and trigger proinflammatory signaling cascades. Our finding that TLR7 expression is upregulated in both epithelial and stromal compartments in human and murine pancreatic cancer led us to postulate that carcinogenesis is dependent on TLR7 signaling. In a mouse model of pancreatic cancer, TLR7 ligation vigorously accelerated tumor progression and induced loss of expression of PTEN, p16, and cyclin D1 and upregulation of p21, p27, p53, c-Myc, SHPTP1, TGF-beta, PPARgamma, and cyclin B1. Furthermore, TLR7 ligation induced STAT3 activation and interfaced with Notch as well as canonical NF-kappaB and MAP kinase pathways, but downregulated expression of Notch target genes. Moreover, blockade of TLR7 protected against carcinogenesis. Since pancreatic tumorigenesis requires stromal expansion, we proposed that TLR7 ligation modulates pancreatic cancer by driving stromal inflammation. Accordingly, we found that mice lacking TLR7 exclusively within their inflammatory cells were protected from neoplasia. These data suggest that targeting TLR7 holds promise for treatment of human pancreatic cancer.

Vitiligo is characterized by depigmented skin patches caused by loss of epidermal melanocytes. Oxidative stress may have a role in vitiligo onset, while autoimmunity contributes to disease progression. In this study, we sought to identify mechanisms that link disease triggers and spreading of lesions. A hallmark of melanocytes at the periphery of vitiligo lesions is dilation of the endoplasmic reticulum (ER). We hypothesized that oxidative stress results in redox disruptions that extend to the ER, causing accumulation of misfolded peptides, which activates the unfolded protein response (UPR). We used 4-tertiary butyl phenol and monobenzyl ether of hydroquinone, known triggers of vitiligo. We show that expression of key UPR components, including the transcription factor X-box-binding protein 1 (XBP1), is increased following exposure of melanocytes to phenols. XBP1 activation increases production of immune mediators IL6 and IL8. Co-treatment with XBP1 inhibitors reduced IL6 and IL8 production induced by phenols, while overexpression of XBP1 alone increased their expression. Thus, melanocytes themselves produce cytokines associated with activation of an immune response following exposure to chemical triggers of vitiligo. These results expand our understanding of the mechanisms underlying melanocyte loss in vitiligo and pathways linking environmental stressors and autoimmunity.Journal of Investigative Dermatology advance online publication, 14 June 2012; doi:10.1038/jid.2012.181.