Faculty Bibliography

Wnt/beta-catenin signaling is a central regulator of adult stem cells. Variable sensitivity of Wnt reporter transgenes, beta-catenin's dual roles in adhesion and signaling, and hair follicle degradation and inflammation resulting from broad deletion of epithelial beta-catenin have precluded clear understanding of Wnt/beta-catenin's functions in adult skin stem cells. By inducibly deleting beta-catenin globally in skin epithelia, only in hair follicle stem cells, or only in interfollicular epidermis and comparing the phenotypes with those caused by ectopic expression of the Wnt/beta-catenin inhibitor Dkk1, we show that this pathway is necessary for hair follicle stem cell proliferation. However, beta-catenin is not required within hair follicle stem cells for their maintenance, and follicles resume proliferating after ectopic Dkk1 has been removed, indicating persistence of functional progenitors. We further unexpectedly discovered a broader role for Wnt/beta-catenin signaling in contributing to progenitor cell proliferation in nonhairy epithelia and interfollicular epidermis under homeostatic, but not inflammatory, conditions.

Clostridium botulinum neurotoxin (BoNT) is a multi-domain protein made up of the approximately 100 kDa heavy chain (HC) and the approximately 50 kDa light chain (LC). The HC can be further subdivided into two halves: the N-terminal translocation domain (TD) and the C-terminal Receptor Binding Domain (RBD). We have investigated the minimal requirements for channel activity and LC translocation. We utilize a cellular protection assay and a single channel/single molecule LC translocation assay to characterize in real time the channel and chaperone activities of BoNT/A truncation constructs in Neuro 2A cells. The unstructured, elongated belt region of the TD is demonstrated to be dispensable for channel activity, although may be required for productive LC translocation. We show that the RBD is not necessary for channel activity or LC translocation, however it dictates the pH threshold of channel insertion into the membrane. These findings indicate that each domain functions as a chaperone for the others in addition to their individual functions, working in concert to achieve productive intoxication.

Previously, we showed an inverse correlation between HSP27 serum levels and experimental atherogenesis in ApoE(-/-) mice that over-express HSP27 and speculated that the apparent binding of HSP27 to scavenger receptor-A (SR-A) was of mechanistic importance in attenuating foam cell formation. However, the nature and importance of the interplay between HSP27 and SR-A in atheroprotection remained unclear. Treatment of THP-1 macrophages with recombinant HSP27 (rHSP27) inhibited acLDL binding (-34%; p<0.005) and uptake (-38%, p<0.05). rHSP27 reduced SR-A mRNA (-39%, p=0.02), total protein (-56%, p=0.01) and cell surface (-53%, p<0.001) expression. The reduction in SR-A expression by rHSP27 was associated with a 4-fold increase in nuclear factor-kappa B (NF-κB) signaling (p<0.001 versus control), while an inhibitor of NF-κB signaling, BAY11-7082, attenuated the negative effects of rHSP27 on both SR-A expression and lipid uptake. To determine if SR-A is required for HSP27 mediated atheroprotection in vivo, ApoE(-/-) and ApoE(-/-) SR-A(-/-) mice fed with a high fat diet were treated for 3weeks with rHSP25. Compared to controls, rHSP25 therapy reduced aortic en face and aortic sinus atherosclerotic lesion size in ApoE(-/-) mice by 39% and 36% (p<0.05), respectively, but not in ApoE(-/-)SR-A(-/-) mice. In conclusion, rHSP27 diminishes SR-A expression, resulting in attenuated foam cell formation in vitro. Regulation of SR-A by HSP27 may involve the participation of NF-κB signaling. Lastly, SR-A is required for HSP27-mediated atheroprotection in vivo.

High Density Lipoprotein (HDL) is a natural nanoparticle involved in the transport of cholesterol throughout the body. HDL has been shown to exhibit atheroprotective properties as it promotes cholesterol efflux from atherosclerotic plaque macrophages in the arterial wall. Various laboratories have focused on the reconstitution of HDL (rHDL) for a variety of reasons, ranging from a better understanding of the structural biology of apolipoproteins to the use of rHDL as an injectable therapeutic (1). A recent effort centers around the use of rHDL as a natural nanoparticle platform for the delivery of contrast agents such as gadolinium chelates, iron oxide or gold nanoparticles, and employing them as molecular imaging contrast agents (2). To date, multistep production protocols pose a limit on the synthesis of batch quantities and are sensitive to inter-batch variations. In order to scale up the production process and to judiciously control rHDL's composition we have developed a modular single-step approach based on recently introduced microfluidics technology (3) that enables the standardized mass production of such lipoprotein-based nanoparticles. Materials and methods Organic solutions containing phospholipids and imaging agents (QD, FeO-NP, Au-NP, DiO) were injected into a microfluidic chip alongside an aqueous solution containing ApoA1. Within the chip the controlled flow streams generate microvortices where fast mixing of the solutions leads to the instantaneous formation of HDL-like nanoparticles (Figure 1). HDL particles produced by this microfluidics method, which we refer to as muHDL, had similar physicochemical properties (size, morphology) to particles produced by conventional methods and natural HDL. Moreover cell based assays demonstrated that muHDL nanoparticles displayed a similar bioactivity profile to natural HDL. muHDL that encapsulated hydrophobic dies (DiO) or nanocrystals such as quantum dots (QD), gold (Au) or iron oxide (FeO) nanoparticles were characterized and evaluated in!

Rhomboid proteases are a family of integral membrane proteins that have been implicated in critical regulatory roles in a wide array of cellular processes and signaling events. The determination of crystal structures of the prokaryotic rhomboid GlpG from Escherichia coli and Haemophilus influenzae has ushered in an era of unprecedented understanding into molecular aspects of intramembrane proteolysis by this fascinating class of protein. A combination of structural studies by X-ray crystallography, and biophysical and spectroscopic analyses, combined with traditional enzymatic and functional analysis has revealed fundamental aspects of rhomboid structure, substrate recognition and the catalytic mechanism. This review summarizes these remarkable advances by examining evidence for the proposed catalytic mechanism derived from inhibitor co-crystal structures, conflicting models of rhomboid-substrate interaction, and recent work on the structure and function of rhomboid cytosolic domains. In addition to exploring progress on aspects of rhomboid structure, areas for future research and unaddressed questions are emphasized and highlighted. This article is part of a Special Issue entitled: Intramembrane Proteases.

Accumulating evidence suggests that epigenetic alterations in brain-derived neurotrophic factor (BDNF) promoters are associated with the pathophysiology of psychiatric disorders. Epigenetic changes in BDNF were reported not only in brain tissues but also in other tissues, including peripheral blood cells (PBC) and saliva. We examined DNA methylation levels of BDNF promoters I and IV using genomic DNA derived from PBC of healthy controls (n=100), and patients with schizophrenia (n=100), all from the Japanese population, by pyrosequencing. The examined CpG sites were chosen based on previous epigenetic studies that reported altered DNA methylation. We found a significantly higher level of methylation at BDNF promoter I in patients with schizophrenia compared to controls, although the difference was small. Subsequent analysis revealed that in controls, the methylation level of BDNF promoters was associated with sex, and the methylation difference observed in promoter I was more prominent in male patients with schizophrenia. Epigenetic alteration of BDNF in the PBC might reflect the pathophysiology of schizophrenia, and could be a potential biomarker.

OBJECTIVE: ANXA6, the gene for annexin A6, is highly expressed in osteoarthritic (OA) articular chondrocytes but not in healthy articular chondrocytes. This study was undertaken to determine whether annexin A6 affects catabolic events in these cells. METHODS: Articular chondrocytes were isolated from Anxa6-knockout mice, wild-type (WT) mice, and human articular cartilage in which ANXA6 was overexpressed. Cells were treated with interleukin-1beta (IL-1beta) or tumor necrosis factor alpha (TNFalpha), and expression of catabolic genes and activation of NF-kappaB were determined by real-time polymerase chain reaction and luciferase reporter assay. Anxa6(-/-) and WT mouse knee joints were injected with IL-1beta or the medial collateral ligament was transected and partial resection of the medial meniscus was performed to determine the role of Anxa6 in IL-1beta-mediated cartilage destruction and OA progression. The mechanism by which Anxa6 stimulates NF-kappaB activity was determined by coimmunoprecipitation and immunoblot analysis of nuclear and cytoplasmic fractions of IL-1beta-treated Anxa6(-/-) and WT mouse chondrocytes for p65 and Anxa6. RESULTS: Loss of Anxa6 resulted in decreased NF-kappaB activation and catabolic marker messenger RNA (mRNA) levels in IL-1beta- or TNFalpha-treated articular chondrocytes, whereas overexpression of ANXA6 resulted in increased NF-kappaB activity and catabolic marker mRNA levels. Annexin A6 interacted with p65, and loss of Anxa6 caused decreased nuclear translocation and retention of the active p50/p65 NF-kappaB complex. Cartilage destruction in Anxa6(-/-) mouse knee joints after IL-1beta injection or partial medial meniscectomy was reduced as compared to that in WT mouse joints. CONCLUSION: Our data define a role of annexin A6 in the modulation of NF-kappaB activity and in the stimulation of catabolic events in articular chondrocytes.