Faculty Bibliography

Human Med26 was originally purified in the Cofactor Required for Sp1 Activation complex (CRSP) as a 70 kilodalton component named CRSP70. This polypeptide was specific to metazoans and the "small" form of the Mediator complex. We report here that a Drosophila homologue of Med26 similarly interacts with other components of the core Drosophila Mediator complex but not with the kinase module, and is recruited to genes upon activation. Using a null allele of Med26, we show that Med26 is required for organismal viability but not for cell proliferation or survival. Clones lacking Med26 in the wing disc lead to loss of the adult wing margin and reduced expression of genes involved in wing margin formation. Surprisingly, when polytene chromosomes from the salivary gland were examined using antibodies to Med26, it was apparent that a fraction of the protein is associated with the chromocenter, which contains pericentric heterochromatin. This staining co-localizes with heterochromatin protein 1 (HP1). Immunoprecipitation experiments show that Med26 interacts with HP1. The interaction is mediated through the chromoshadow domain of HP1 and through the conserved motif in the carboxy-terminus of the Med26 protein. This work is the first characterization of the metazoan-specific Mediator subunit in an animal model.

Amphotericin B remains the preferred choice for leishmanial infection, but it has limited clinical applications due to substantial dose limiting toxicities. In the present work, AmB has been formulated in lipo-polymerosome (L-Psome) by spontaneous self-assembly of synthesized glycol chitosan-stearic acid copolymer. The optimized L-Psome formulation with vesicle size of 243.5 +/- 17.9 nm, PDI of 0.168 +/- 0.08 and zeta potential of (+) 27.15 +/- 0.46 mV with 25.59 +/- 0.87% AmB loading was obtained. The field emission scanning electron microscopy (FESEM) and high resolution transmission electron microscopy (HRTEM) images suggest nearly spherical morphology of L-Psome. An in vitro study showed comparatively sustained AmB release (66.082 +/- 1.73% within 24 h) and high plasma stability compared to commercial Ambisome and Fungizone, where glycol chitosan content was found to be efficient in preventing L-Psome destabilization in the presence of plasma protein. In vitro and in vivo toxicity studies revealed less toxicity of AmB-L-Psome compared to commercialized Fungizone and Ambisome favored by monomeric form of AmB within L-Psome, observed by UV-visible spectroscopy. Experimental results of in vitro (macrophage amastigote system) and in vivo (Leishmania donovani infected hamsters) illustrated the efficacy of AmB-L-Psome to augment effective antileishmanial properties supported by upregulation of Th-1 cytokines (TNF-alpha, IL-12 and IFN-gamma) and inducible nitric oxide synthase, and downregulation of Th-2 cytokines (TGF-beta, IL-10 and IL-4), measured by quantitative mRNA analysis by real time PCR (RT-PCR). Conclusively, developed L-Psome system could be a viable alternative to the current less stable, toxic commercial formulations and developed as a highly efficacious drug delivery system.

Nrf2:INrf2 (Keap1) are cellular sensors of oxidative and electrophilic stress. Nrf2 is a nuclear factor that controls the expression and coordinated induction of a battery of genes that encode detoxifying enzymes, drug transporters, antiapoptotic proteins, and proteasomes. In the basal state, Nrf2 is constantly degraded in the cytoplasm by its inhibitor, INrf2. INrf2 functions as an adapter for Cul3/Rbx1 E3 ubiquitin ligase-mediated degradation of Nrf2. Chemicals, including antioxidants, tocopherols including alpha-tocopherol (vitamin E), and phytochemicals, and radiation antagonize the Nrf2:INrf2 interaction and lead to the stabilization and activation of Nrf2. The signaling events involve preinduction, induction, and postinduction responses that tightly control Nrf2 activation and repression back to the basal state. Oxidative/electrophilic signals activate unknown tyrosine kinases in a preinduction response that phosphorylates specific residues on Nrf2 negative regulators, INrf2, Fyn, and Bach1, leading to their nuclear export, ubiquitination, and degradation. This prepares nuclei for unhindered import of Nrf2. Oxidative/electrophilic modification of INrf2 cysteine 151 followed by PKC phosphorylation of Nrf2 serine 40 in the induction response results in the escape or release of Nrf2 from INrf2. Nrf2 is thus stabilized and translocates to the nucleus, resulting in a coordinated activation of gene expression. This is followed by a postinduction response that controls the "switching off" of Nrf2-activated gene expression. GSK3beta, under the control of AKT and PI3K, phosphorylates Fyn, leading to Fyn nuclear localization. Fyn phosphorylates Nrf2 Y568, resulting in nuclear export and degradation of Nrf2. The activation and repression of Nrf2 provide protection against oxidative/electrophilic stress and associated diseases, including cancer. However, deregulation of INrf2 and Nrf2 due to mutations may lead to nuclear accumulation of Nrf2 that reduces apoptosis and promotes oncogenesis and drug resistance.

The phosphoinositide-3 kinase (PI3K) pathway is deregulated in a significant proportion of melanomas, and PI3K pathway activation in combination with constitutively active mitogen-activated protein kinase signaling shows synergistic effects in the process of melanoma tumorigenesis. Recently, a tumor suppressor function for the lipid phosphatase inositol polyphosphate 4-phosphatase type II (INPP4B) has been described in breast and prostate cancers, with impact on PI3K signaling output. Given the importance of PI3K pathway activity for melanoma formation and growth, we aimed to assess the role of INPP4B in melanocytic tumors. Our studies in native tumors suggest that decreased INPP4B expression is an event correlating with tumor progression in melanocytic neoplasms. We further demonstrate that INPP4B regulates PI3K/Akt signaling and exerts a tumor suppressor effect, impacting the proliferative, invasive, and tumorigenic capacity of melanoma cells. INPP4B expression in melanocytic neoplasms may therefore have potential as a biomarker for disease progression and as a modulator for the prediction of treatment outcome.

Previously, we showed that discoidin domain receptor 1 (DDR1), a class of collagen-activated receptor tyrosine kinase (RTK) was highly upregulated on bone marrow (BM)-derived CD33+ leukemic blasts of acute myeloid leukemia (AML) patients. Herein as DDR1 is a class of collagen-activated RTK, we attempt to understand the role of native and remodeled collagen IV in BM microenvironment and its functional significance in leukemic cells. Exposure to denatured collagen IV significantly increased the migration and adhesion of K562 cells, which also resulted in increased activation of DDR1 and AKT. Further, levels of MMP9 were increased in conditioned media (CM) of denatured collagen IV exposed cells. Mass spectrometric liquid chromatography/tandem mass spectrometry QSTAR proteomic analysis revealed exclusive presence of Secretogranin 3 and InaD-like protein in the denatured collagen IV CM. Importantly, BM samples of AML patients exhibited increased levels of remodeled collagen IV compared to native as analyzed via anti-HUIV26 antibody. Taken together, for the first time, we demonstrate that remodeled collagen IV is a potent activator of DDR1 and AKT that also modulates both migration and adhesion of myeloid leukemia cells. Additionally, high levels of the HUIV26 cryptic collagen IV epitope are expressed in BM of AML patients. Further understanding of this phenomenon may lead to the development of therapeutic agents that directly modulate the BM microenvironment and attenuate leukemogenesis.

Noncoding RNAs (ncRNAs) represent a class of RNA molecules that typically do not code for proteins. Emerging data suggest that ncRNAs play an important role in several physiological and pathological conditions such as cancer and cardiovascular diseases, including atherosclerosis. The best-characterized ncRNAs are the microRNAs which are small, approximately 22-nucleotide sequences of RNA that regulate gene expression at the posttranscriptional level through transcript degradation or translational repression. MicroRNAs control several aspects of atherosclerosis, including endothelial cell, vascular smooth cell, and macrophage functions as well as lipoprotein metabolism. Apart from microRNAs, recently ncRNAs, especially long ncRNAs, have emerged as important potential regulators of the progression of atherosclerosis. However, the molecular mechanism of their regulation and function as well as the significance of other ncRNAs such as small nucleolar RNAs during atherogenesis is largely unknown. In this review, we summarize the recent findings in the field, highlighting the importance of ncRNAs in atherosclerosis and discuss their potential use as therapeutic targets in cardiovascular diseases.

The ATP-binding cassette transporter A1 (ABCA1) is a major regulator of cellular cholesterol efflux and plasma high-density lipoprotein (HDL) biogenesis. Even though the transcriptional activation of ABCA1 is well established, the post-transcriptional regulation of ABCA1 expression is poorly understood. Here, we investigate the potential contribution of the Ribonucleoprotein-binding protein (RIP), HuR on the post-transcriptional regulation of ABCA1 expression. RNA immunoprecipitation assays demonstrate a direct interaction between HuR and ABCA1 mRNA. We found that HuR binds to the 3-untranslated region (UTR) region of ABCA1 and increases ABCA1 translation, while HuR silencing reduces ABCA1 expression and cholesterol efflux to ApoA1 in human hepatic (Huh-7) and monocytic (THP-1) cells. Interestingly, cellular cholesterol levels regulate the expression, intracellular localization and interaction between HuR and ABCA1 mRNA. Finally, we found that HuR expression was significantly increased in macrophages from human atherosclerotic plaques, suggesting an important role for this RBP in controlling macrophage cholesterol metabolism in vivo. In summary, we have identified HuR as a novel post-transcriptional regulator of ABCA1 expression and cellular cholesterol homeostasis, thereby opening new avenues for increasing cholesterol efflux from atherosclerotic foam macrophages and raising circulating HDL cholesterol levels.

Atherosclerosis is a pathologic condition caused by chronic inflammation in response to lipid deposition in the arterial wall. There are many known contributing factors such as long-term abnormal glucose levels, smoking, hypertension, and hyperlipidemia. Under the influence of such factors, immune and non-immune effectors cells are activated and participate during the progression of atherosclerosis. Protein kinase C (PKC) family isoforms are key players in the signal transduction pathways of cellular activation and have been associated with several aspects of the atherosclerotic vascular disease. This review article summarizes the current knowledge of PKC isoforms functions during atherogenesis, and addresses differential roles and disputable observations of PKC isoforms. Among PKC isoforms, both PKCbeta and PKCdelta are the most attractive and potential therapeutic targets. This commentary discusses in detail the outcomes and current status of clinical trials on PKCbeta and PKCdelta inhibitors in atherosclerosis-associated disorders like diabetes and myocardial infarction. The risk and benefit of these inhibitors for clinical purposes will be also discussed. This review summarizes what is already being done and what else needs to be done in further targeting PKC isoforms, especially PKCbeta and PKCdelta, for therapy of atherosclerosis and atherosclerosis-associated vasculopathies in the future.