Faculty Bibliography

Diabetic wounds are a significant public health burden, with slow or nonhealing diabetic foot ulcers representing the leading cause of non-traumatic lower limb amputation in developed countries. These wounds heal poorly as a result of compromised blood vessel formation in response to ischemia. We have recently shown that this impairment in neovascularization results from a high glucose-induced defect in transactivation of hypoxia-inducible factor-1alpha (HIF-1alpha), the transcription factor regulating vascular endothelial growth factor (VEGF) expression. HIF-1 dysfunction is the end result of reactive oxygen species-induced modification of its coactivator p300 by the glycolytic metabolite methylglyoxal. Use of the iron chelator-antioxidant deferoxamine (DFO) reversed these effects and normalized healing of humanized diabetic wounds in mice. Here, we present additional data demonstrating that HIF-1alpha activity, not stability, is impaired in the high glucose environment. We demonstrate that high glucose-induced impairments in HIF-1alpha transactivation persist even in the setting of constitutive HIF-1alpha protein overexpression. Further, we show that high glucose-induced hydroxylation of the C-terminal transactivation domain of HIF-1alpha (the primary pathway regulating HIF-1alpha/p300 binding) does not alter HIF-1alpha activity. We extend our study of DFO's therapeutic efficacy in the treatment of impaired wound healing by demonstrating improvements in tissue viability in diabetic mice with DFO-induced increases in VEGF expression and vascular proliferation. Since DFO has been in clinical use for decades, the potential of this drug to treat a variety of ischemic conditions in humans can be evaluated relatively quickly.

The regulated migration of cells is essential for development and tissue homeostasis, and aberrant cell migration can lead to an impaired immune response and the progression of cancer. Primordial germ cells (PGCs), precursors to sperm and eggs, have to migrate across the embryo to reach somatic gonadal precursors, where they carry out their function. Studies of model organisms have revealed that, despite important differences, several features of PGC migration are conserved. PGCs require an intrinsic motility programme and external guidance cues to survive and successfully migrate. Proper guidance involves both attractive and repulsive cues and is mediated by protein and lipid signalling

Cell membrane proteins play an important role in tissue architecture and cell-cell communication. We hypothesize that segmentation and multidimensional characterization of the distribution of cell membrane proteins, on a cell-by-cell basis, enable improved classification of treatment groups and identify important characteristics that can otherwise be hidden. We have developed a series of computational steps to 1) delineate cell membrane protein signals and associate them with a specific nucleus; 2) compute a coupled representation of the multiplexed DNA content with membrane proteins; 3) rank computed features associated with such a multidimensional representation; 4) visualize selected features for comparative evaluation through heatmaps; and 5) discriminate between treatment groups in an optimal fashion. The novelty of our method is in the segmentation of the membrane signal and the multidimensional representation of phenotypic signature on a cell-by-cell basis. To test the utility of this method, the proposed computational steps were applied to images of cells that have been irradiated with different radiation qualities in the presence and absence of other small molecules. These samples are labeled for their DNA content and E-cadherin membrane proteins. We demonstrate that multidimensional representations of cell-by-cell phenotypes improve predictive and visualization capabilities among different treatment groups, and identify hidden variables

Tyrosine hydroxylase (TH) promoter activity is induced by 17beta-estradiol (E(2)) in PC12 cells expressing estradiol receptor-alpha (ERalpha) requiring a cAMP/calcium response element (CRE/CaRE) at -45. To examine whether membrane-initiated estradiol signaling is underlying this induction, cells co-transfected with TH reporter construct and ERalpha expression vector were exposed to membrane-impermeant estradiol conjugate (beta-estradiol-6-(O-carboxy-methyl) oxime-bovine serum albumin, E(2)BSA). TH promoter activity was elevated by E(2)BSA in dose- and time-dependent manner. E(2)BSA also elicited rapid phosphorylation of CRE binding protein (CREB) and increased CRE-driven promoter activity. Over-expression of dominant negative forms of CREB, with mutations in DNA binding or phosphorylation site, prevented TH promoter response to E(2)BSA. Pre-treatment with protein kinase A (PKA) and MEK inhibitors reduced E(2) dependent phosphorylation of CREB and ERK, and also decreased induction of TH promoter activity by E(2) or E(2)BSA. Blocking S-palmitoylation of ERalpha with C451A mutation and/or pre-treatment with 2-Bromopalmitate did not prevent but instead enhanced E(2) or E(2)BSA-elicited induction of TH promoter activity. These findings reveal, for the first time, that estradiol induction of TH gene transcription with ERalpha in PC12 cells involves membrane-initiated estradiol signaling, rapid activation of dual PKA/MEK signaling pathways, leading to CREB phosphorylation, acting at CRE/CaRE. The data demonstrate possible mechanism whereby estradiol affects catecholaminergic systems in vivo.

Partitioning tissues into compartments that do not intermix is essential for the correct morphogenesis of animal embryos and organs. Several hypotheses have been proposed to explain compartmental cell sorting, mainly differential adhesion, but also regulation of the cytoskeleton or of cell proliferation. Nevertheless, the molecular and cellular mechanisms that keep cells apart at boundaries remain unclear. Here we demonstrate, in early Drosophila melanogaster embryos, that actomyosin-based barriers stop cells from invading neighbouring compartments. Our analysis shows that cells can transiently invade neighbouring compartments, especially when they divide, but are then pushed back into their compartment of origin. Actomyosin cytoskeletal components are enriched at compartmental boundaries, forming cable-like structures when the epidermis is mitotically active. When MyoII (non-muscle myosin II) function is inhibited, including locally at the cable by chromophore-assisted laser inactivation (CALI), in live embryos, dividing cells are no longer pushed back, leading to compartmental cell mixing. We propose that local regulation of actomyosin contractibility, rather than differential adhesion, is the primary mechanism sorting cells at compartmental boundaries.

The derivation of induced pluripotent stem cells (iPSCs) usually involves the viral introduction of reprogramming factors into somatic cells. Here we used gene targeting to generate a mouse strain with a single copy of an inducible, polycistronic reprogramming cassette, allowing for the induction of pluripotency in various somatic cell types. As these 'reprogrammable mice' can be easily bred, they are a useful tool to study the mechanisms underlying cellular reprogramming

The ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family consists of 19 proteases. These enzymes are known to play important roles in development, angiogenesis and coagulation; dysregulation and mutation of these enzymes have been implicated in many disease processes, such as inflammation, cancer, arthritis and atherosclerosis. This review briefly summarizes the structural organization and functional roles of ADAMTSs in normal and pathological conditions, focusing on members that are known to be involved in the degradation of extracellular matrix and loss of cartilage in arthritis, including the aggrecanases (ADAMTS-4 and ADAMTS-5), ADAMTS-7 and ADAMTS-12, the latter two are associated with cartilage oligomeric matrix protein (COMP), a component of the cartilage extracellular matrix (ECM). We will discuss the expression pattern and the regulation of these metalloproteinases at multiple levels, including their interaction with substrates, induction by pro-inflammatory cytokines, protein processing, inhibition (e.g., TIMP-3, alpha-2-macroglobulin, GEP), and activation (e.g., syndecan-4, PACE-4)

Several lines of evidence point to the central role of WNT signaling in the initiation of intestinal tumorigenesis, most often due to loss of APC, a negative regulator of the WNT-betaCATENIN/TCF pathway. Modeling human colon cancers in mice through loss of Apc has shown that inappropriate activation of Wnt signaling is sufficient to induce adenoma formation. More recent analyses have also demonstrated a key role for HEDGEHOG-GLI (HH-GLI) signaling in human colon cancers. However, how the WNT and HH pathways interact during intestinal development, homeostasis and cancer is not clear. Marker analyses suggest predominant paracrine signaling from rare Shh producing cells in the crypt's bottom to adjacent Gli1(+) mesenchymal cells in normal adult mice. Using conditional KO models, we show that inhibition of the function of the critical Hh mediator Smoothened (Smo) rescues the lethality and intestinal phenotypes of loss of Apc. The results uncover an essential role of the Hh pathway in tumors induced by hyperactive Wnt signaling, suggest the action of the Hh pathway in parallel or downstream of Wnt signaling, and validate this model for its use in preclinical work testing Hh pathway antagonists.

An 11-year-old girl with a history of diabetes mellitus type I and celiac disease presented with multiple, depressed patches of purple-brown skin on the right lower extremity and central back, with histopathologic features of early morphea. Though morphea may coexist with other autoimmune diseases, its presentation with both diabetes mellitus type I and celiac disease has not yet been described

Development involves the establishment of boundaries between fields specified to differentiate into distinct tissues. The Drosophila larval eye-antennal imaginal disc must be subdivided into regions that differentiate into the adult eye, antenna and head cuticle. We have found that the transcriptional co-factor Chip is required for cells at the ventral eye-antennal disc border to take on a head cuticle fate; clones of Chip mutant cells in this region instead form outgrowths that differentiate into ectopic eye tissue. Chip acts independently of the transcription factor Homothorax, which was previously shown to promote head cuticle development in the same region. Chip and its vertebrate CLIM homologues have been shown to form complexes with LIM-homeodomain transcription factors, and the domain of Chip that mediates these interactions is required for its ability to suppress the eye fate. We show that two LIM-homeodomain proteins, Arrowhead and Lim1, are expressed in the region of the eye-antennal disc affected in Chip mutants, and that both require Chip for their ability to suppress photoreceptor differentiation when misexpressed in the eye field. Loss-of-function studies support the model that Arrowhead and Lim1 act redundantly, using Chip as a co-factor, to prevent retinal differentiation in regions of the eye disc destined to become ventral head tissue