Faculty Bibliography

The catheter-associated uropathogenProteus mirabilisfrequently causes urinary stones, but little has been known about the initial stages of bladder colonization and stone formation. We found thatP. mirabilisrapidly invades the bladder urothelium, but generally fails to establish an intracellular niche. Instead, it forms extracellular clusters in the bladder lumen, which form foci of mineral deposition consistent with development of urinary stones. These clusters elicit a robust neutrophil response, and we present evidence of neutrophil extracellular trap generation during experimental urinary tract infection. We identified two virulence factors required for cluster development: urease, which is required for urolithiasis, and mannose-resistantProteus-like fimbriae. The extracellular cluster formation byP. mirabilisstands in direct contrast to uropathogenicEscherichia coli, which readily formed intracellular bacterial communities but not luminal clusters or urinary stones. We propose that extracellular clusters are a key mechanism ofP. mirabilissurvival and virulence in the bladder.

AIMS: Myocardial ischemia remains the prime cause of morbidity and mortality in the United States. Ischemic preconditioning (IPC) is a powerful form of endogenous protection against myocardial infarction. We studied alterations in KATPchannels surface density as a potential mechanism of IPC's protection. METHODS AND RESULTS: Using cardiac-specific knockout of Kir6.2 subunits, we demonstrate an essential role for sarcolemmal KATPchannels in the infarct-limiting effect of IPC in the mouse heart. With biochemical membrane fractionation, we demonstrated that sarcolemmal KATPchannel subunits are distributed both to the sarcolemma and intracellular endosomal compartments. Global ischemia causes a loss of sarcolemmal KATPchannel subunit distribution and internalization to endosomal compartments. Ischemia-induced internalization of KATPchannels was prevented by CaMKII inhibition. KATPchannel subcellular redistribution was also observed with immunohistochemistry. Ischemic preconditioning prior to the index ischemia reduces not only the infarct size, but also prevents KATPchannel internalization. Furthermore, not only did adenosine mimic IPC by preventing infarct size, but it also prevented ischemia-induced KATPchannel internalization via a PKC-mediated pathway. We show that preventing endocytosis with dynasore reduces both KATPchannel internalization and strongly mitigates infarct development. CONCLUSIONS: Our data demonstrate that plasticity of KATPchannel surface expression must be considered as a potentially important mechanism of the protective effects of IPC and adenosine.

Liver X receptors (LXR) are oxysterol-activated nuclear receptors that play a central role in reverse cholesterol transport (RCT) through upregulation of ATP-binding Cassette transporters (ABCA1 and ABCG1) that mediate cellular cholesterol efflux. Mouse models of atherosclerosis exhibit reduced atherosclerosis and enhanced regression of established plaques upon LXR activation. However, the coregulatory factors that affect LXR-dependent gene activation in macrophages remain to be elucidated. To identify novel regulators of LXR that modulate its activity, we used affinity purification and mass spectrometry to analyze nuclear LXRalpha complexes, and identified poly(ADP-ribose) polymerase-1 (PARP-1) as an LXR-associated factor. In fact, PARP-1 interacted with both LXRalpha and LXRbeta. Both depletion of PARP-1 and inhibition of PARP-1 activity augmented LXR ligand-induced ABCA1 expression in the RAW 264.7 macrophage line and primary bone marrow derived macrophages, but did not affect LXR-dependent expression of other target genes, ABCG1 and SREBP-1c. Chromatin immunoprecipitation experiments confirmed PARP-1 recruitment at the LXR response element in the promoter of the ABCA1 gene. Further, we demonstrated that LXR is poly(ADP-ribosyl)ated by PARP-1, a potential mechanism by which PARP-1 influences LXR function. Importantly, the PARP inhibitor, 3-aminobenzamide, enhanced macrophage ABCA1-mediated cholesterol efflux to the lipid-poor apolipoprotein AI (apoA-I). These findings shed light on the important role of PARP-1 on LXR-regulated lipid homeostasis. Understanding the interplay between PARP-1 and LXR may provide insights into developing novel therapeutics for treating atherosclerosis.

Clear mechanistic understanding of the biological processes elicited by radiation that increase cancer risk can be used to inform prediction of health consequences of medical uses, such as radiotherapy, or occupational exposures, such as those of astronauts during deep space travel. Here, we review the current concepts of carcinogenesis as a multicellular process during which transformed cells escape normal tissue controls, including the immune system, and establish a tumor microenvironment. We discuss the contribution of two broad classes of radiation effects that may increase cancer: radiation targeted effects that occur as a result of direct energy deposition, e.g., DNA damage, and non-targeted effects (NTE) that result from changes in cell signaling, e.g., genomic instability. It is unknown whether the potentially greater carcinogenic effect of high Z and energy (HZE) particle radiation is a function of the relative contribution or extent of NTE or due to unique NTE. We addressed this problem using a radiation/genetic mammary chimera mouse model of breast cancer. Our experiments suggest that NTE promote more aggressive cancers, as evidenced by increased growth rate, transcriptomic signatures, and metastasis, and that HZE particle NTE are more effective than reference gamma-radiation. Emerging evidence suggest that HZE irradiation dampens antitumor immunity. These studies raise concern that HZE radiation exposure not only increases the likelihood of developing cancer but also could promote progression to more aggressive cancer with a greater risk of mortality.

Uroplakins (UPs) are major differentiation products of urothelial umbrella cells, playing important roles in forming the permeability barrier, and in the expansion/stabilization of the apical membrane. Further, UPIa serves as a uropathogenic E. coli receptor. While it is understood that UPs are delivered to the apical membrane via fusiform vesicles (FVs), the mechanisms that regulate this exocytic pathway remain poorly understood. Immuno-microscopy of normal and mutant mouse urothelia showed that the UP-delivering FVs contained Rab8/11 and Rab27b/Slac2-a, which mediate apical transport along actin filaments. Subsequently, a Rab27b/Slp2-a complex mediated FV-membrane anchorage before SNARE-mediated and MAL-facilitated apical fusion. We also showed that keratin 20 (K20), which formed a chicken-wire network 150-300 nm below the apical membrane and had hole sizes allowing FV passage, defined a subapical compartment containing FVs primed and strategically located for fusion. Finally, we showed that Rab8/11 and Rab27b function in the same pathway, that Rab27b-knockout leads to uroplakin and Slp2-a destabilization, and that Rab27b works upstream from MAL. These data support a unifying model in which UP cargoes are targeted for apical insertion via sequential interactions with Rabs and their effectors, SNAREs and MAL, and in which K20 plays a key role in regulating vesicular trafficking.

BACKGROUND: Reconstruction of soft tissue defects has traditionally relied on the use of grafts and flaps, which may be associated with variable resorption and/or significant donor site morbidity. Cell-based strategies employing adipose-derived stromal cells (ASCs), found within the stromal vascular fraction (SVF) of adipose tissue, may offer an alternative strategy for soft tissue reconstruction. In this study, we investigated the potential of a bone morphogenetic protein receptor type 1A (BMPR1A)(+) subpopulation of ASCs to enhance de novo adipogenesis. METHODS: Human lipoaspirate was enzymatically digested to isolate SVF and magnetic-activated cell separation was utilized to obtain BMPR1A(+) and BMPR1A(-) cells. These cells, along with unenriched cells, were expanded in culture and evaluated for adipogenic gene expression and in vitro adipocyte formation. Cells from each group were also labeled with a green fluorescent protein (GFP) lentivirus and transplanted into the inguinal fat pads, an adipogenic niche, of immunocompromised mice to determine their potential for de novo adipogenesis. Confocal microscopy along with staining of lipid droplets and vasculature was performed to evaluate the formation of mature adipocytes by transplanted cells. RESULTS: In comparison to BMPR1A(-) and unenriched ASCs, BMPR1A(+) cells demonstrated significantly enhanced adipogenesis when cultured in an adipogenic differentiation medium, as evidenced by increased staining with Oil Red O and increased expression of peroxisome proliferator-activating receptor gamma (PPAR-gamma) and fatty acid-binding protein 4 (FABP4). BMPR1A(+) cells also formed significantly more adipocytes in vivo, as demonstrated by quantification of GFP+ adipocytes. Minimal formation of mature adipocytes was appreciated by BMPR1A(-) cells. CONCLUSIONS: BMPR1A(+) ASCs show an enhanced ability for adipogenesis in vitro, as shown by gene expression and histological staining. Furthermore, within an adipogenic niche, BMPR1A(+) cells possessed an increased capacity to generate de novo fat compared to BMPR1A(-) and unenriched cells. This suggests utility for the BMPR1A(+) subpopulation in cell-based strategies for soft tissue reconstruction.

Stem cells are classified into embryonic stem cells and adult stem cells. An evolving alternative to conventional stem cell therapies is induced pluripotent stem cells (iPSCs), which have a multi-lineage potential comparable to conventionally acquired embryonic stem cells with the additional benefits of being less immunoreactive and avoiding many of the ethical concerns raised with the use of embryonic material. The ability to generate iPSCs from somatic cells provides tremendous promise for regenerative medicine. The breakthrough of iPSCs has raised the possibility that patient-specific iPSCs can provide autologous cells for cell therapy without the concern for immune rejection. iPSCs are also relevant tools for modeling human diseases and drugs screening. However, there are still several hurdles to overcome before iPSCs can be used for translational purposes. Here, we review the recent advances in somatic reprogramming and the challenges that must be overcome to move this strategy closer to clinical application.

Obesity and metabolic disorders are a major health concern in all developed countries and a primary focus of current medical research is to improve our understanding treatment of metabolic diseases. One avenue of research that has attracted a great deal of recent interest focuses upon understanding the role of miRNAs in the development of metabolic diseases. miRNAs have been shown to be dysregulated in a number of different tissues under conditions of obesity and insulin resistance, and have been demonstrated to be important regulators of a number of critical metabolic functions, including insulin secretion in the pancreas, lipid and glucose metabolism in the liver, and nutrient signaling in the hypothalamus. In this review we will focus on the important role of miRNAs in regulating the differentiation and function of white and brown adipose tissue and the potential importance of this for maintaining metabolic function and treating metabolic diseases. This article is part of a Special Issue entitled: MicroRNAs and lipid/energy metabolism and related diseases edited by Carlos Fernandez-Hernando and Yajaira Suarez.

KCNQ (voltage-gated K(+) channel family 7 (Kv7)) channels control cellular excitability and underlie the K(+) current sensitive to muscarinic receptor signaling (the M current) in sympathetic neurons. Here we show that the novel anti-epileptic drug retigabine (RTG) modulates channel function of pore-only modules (PMs) of the human Kv7.2 and Kv7.3 homomeric channels and of Kv7.2/3 heteromeric channels by prolonging the residence time in the open state. In addition, the Kv7 channel PMs are shown to recapitulate the single-channel permeation and pharmacological specificity characteristics of the corresponding full-length proteins in their native cellular context. A mutation (W265L) in the reconstituted Kv7.3 PM renders the channel insensitive to RTG and favors the conductive conformation of the PM, in agreement to what is observed when the Kv7.3 mutant is heterologously expressed. On the basis of the new findings and homology models of the closed and open conformations of the Kv7.3 PM, we propose a structural mechanism for the gating of the Kv7.3 PM and for the site of action of RTG as a Kv7.2/Kv7.3 K(+) current activator. The results validate the modular design of human Kv channels and highlight the PM as a high-fidelity target for drug screening of Kv channels.