Faculty Bibliography

BACKGROUND: Melanoma is a heterogeneous tumor with subgroups requiring distinct therapeutic strategies. Genetic dissection of melanoma subgroups and identification of therapeutic agents are of great interest in the field. These efforts will ultimately lead to treatment strategies, likely combinatorial, based on genetic information. METHODS: To identify "driver" genes that can be targeted therapeutically, we screened metastatic melanomas for somatic mutations by exome sequencing followed by selecting those with available targeted therapies directed to the gene product or its functional partner. The FBXW7 gene and its substrate NOTCH1 were identified and further examined. Mutation profiling of FBXW7, biological relevance of these mutations and its inactivation, and pharmacological inhibition of NOTCH1 were examined using in vitro and in vivo assays. RESULTS: We found FBXW7 to be mutated in eight (8.1%) melanoma patients in our cohort (n = 103). Protein expression analysis in human tissue samples (n = 96) and melanoma cell lines (n = 20) showed FBXW7 inactivation as a common event in melanoma (40.0% of cell lines). As a result of FBXW7 loss, we observed an accumulation of its substrates, such as NOTCH1. Ectopic expression of mutant forms of FBXW7 (by 2.4-fold), as well as silencing of FBXW7 in immortalized melanocytes, accelerated tumor formation in vivo (by 3.9-fold). Its inactivation led to NOTCH1 activation, upregulation of NOTCH1 target genes (by 2.6-fold), and promotion of tumor angiogenesis and resulted in tumor shrinkage upon NOTCH1 inhibition (by fivefold). CONCLUSIONS: Our data provides evidence on FBXW7 as a critical tumor suppressor mutated and inactivated in melanoma that results in sustained NOTCH1 activation and renders NOTCH signaling inhibition as a promising therapeutic strategy in this setting.

BACKGROUND: Frostbite injury occurs when exposure to cold results in frozen tissue. We recently reported a novel mouse model for frostbite injury to be used in screening potentially therapeutic drugs and other modalities. OBJECTIVE: We used the mouse skin frostbite model to evaluate the effect of poly-l-arginine contained in lotion (PAL) applied topically to involved skin. METHODS: Sixty mice were studied in a randomized, double-blind method. Standardized 2.9-cm-diameter circles were tattooed on the mouse dorsum. Magnets snap frozen in dry ice (-78.5 degrees C) were used to create a frostbite injury on skin within the circle as a continuous 5-minute freeze. Mice were treated with prefreeze placebo, postthaw placebo, combined prefreeze and postthaw placebo, prefreeze with PAL, postthaw with PAL, or combined prefreeze and postthaw with PAL. Appearance, healing rate, tissue loss, and histology were recorded until the wounds were healed. RESULTS: Application of PAL before inducing frostbite injury resulted in decreased tissue loss as compared with other treatment conditions. CONCLUSIONS: Applying PAL topically to frostbitten mouse skin caused decreased tissue loss. Poly-l-arginine should be studied further to determine whether it is a beneficial therapeutic modality for frostbite injury.

Wound healing is a well-regulated but complex process that involves haemostasis, inflammation, proliferation and maturation. Recent reports suggest that microRNAs (miRs) play important roles in dermal wound healing. In fact, miR deregulation has been linked with impaired wound repair. miR-155 has been shown to be induced by inflammatory mediators and plays a central regulatory role in immune responses. We have investigated the potential role of miR-155 in wound healing. By creating punch wounds in the skin of mice, we found an increased expression of miR-155 in wound tissue when compared with healthy skin. Interestingly, analysis of wounds of mice lacking the expression of miR-155 (miR-155-/- ) revealed an increased wound closure when compared with wild-type animals. Also, the accelerated wound closing correlated with elevated numbers of macrophages in wounded tissue. Gene expression analysis of wounds tissue and macrophages isolated from miR-155-/- mice that were treated with interleukin-4 demonstrated an increased expression of miR-155 targets (BCL6, RhoA and SHIP1) as well as, the finding in inflammatory zone-1 (FIZZ1) gene, when compared with WT mice. Moreover, the up-regulated levels of FIZZ1 in the wound tissue of miR-155-/- mice correlated with an increased deposition of type-1 collagens, a phenomenon known to be beneficial in wound closure. Our data indicate that the absence of miR-155 has beneficial effects in the wound healing process.

Gentamicin (2 and 10 mug/mL) was bactericidal against 64% and 100% of gentamicin-susceptible, KPC-2-producing Klebsiella pneumoniae strains, respectively. Doripenem (8 mug/mL)+colistin (2 mug/mL) was inferior to gentamicin (2 mug/mL), doripenem+gentamicin, gentamicin+colistin or doripenem+gentamicin+colistin against insAA134-135GD ompK36 porin mutants (n=9). Doripenem+colistin was comparable to other 2- or 3-drug regimens and superior to single drugs against wild-type/minor ompK36 mutants (n=5). An algorithm incorporating ompK36 genotypes, and gentamicin and doripenem susceptibility may predict antimicrobial activity against KPC-K. pneumoniae.

Human endogenous retroviruses (HERVs) are viruses that have colonized the germ line and spread through vertical passage. Only the more recently acquired HERVs, such as the HERV-K (HML-2) group, maintain coding open reading frames. Expression of HERV-Ks has been linked to different pathological conditions, including HIV infection, but our knowledge on which specific HERV-Ks are expressed in primary lymphocytes currently is very limited. To identify the most expressed HERV-Ks in an unbiased manner, we analyzed their expression patterns in peripheral blood lymphocytes using Pacific Biosciences (PacBio) single-molecule real-time (SMRT) sequencing. We observe that three HERV-Ks (KII, K102, and K18) constitute over 90% of the total HERV-K expression in primary human lymphocytes of five different donors. We also show experimentally that two of these HERV-K env sequences (K18 and K102) retain their ability to produce full-length and posttranslationally processed envelope proteins in cell culture. We show that HERV-K18 Env can be incorporated into HIV-1 but not simian immunodeficiency virus (SIV) particles. Moreover, HERV-K18 Env incorporation into HIV-1 virions is dependent on HIV-1 matrix. Taken together, we generated high-resolution HERV-K expression profiles specific for activated human lymphocytes. We found that one of the most abundantly expressed HERV-K envelopes not only makes a full-length protein but also specifically interacts with HIV-1. Our findings raise the possibility that these endogenous retroviral Env proteins could directly influence HIV-1 replication. IMPORTANCE: Here, we report the HERV-K expression profile of primary lymphocytes from 5 different healthy donors. We used a novel deep-sequencing technology (PacBio SMRT) that produces the long reads necessary to discriminate the complexity of HERV-K expression. We find that primary lymphocytes express up to 32 different HERV-K envelopes, and that at least two of the most expressed Env proteins retain their ability to make a protein. Importantly, one of them, the envelope glycoprotein of HERV-K18, is incorporated into HIV-1 in an HIV matrix-specific fashion. The ramifications of such interactions are discussed, as the possibility of HIV-1 target tissue broadening and immune evasion are considered.

Abstract Cell Communication and Adhesion has been fortunate to enlist two pioneers of epidermal and cardiac cell junctions, Kathleen Green and Mario Delmar, as Guest Editors of a two part series on junctional targets of skin and heart disease. Part 2 of this series begins with an overview from Dipal Patel and Kathy Green comparing epidermal desmosomes to cardiac area composita junctions, and surveying the pathogenic mechanisms resulting from mutations in their components in heart disease. This is followed by a review from David Kelsell on the role of desmosomal mutation in inherited syndromes involving skin fragility. Agnieszka Kobeliak discusses how structural deficits in the epidermal barrier intersect with the NFkB signaling pathway to induce inflammatory diseases such as psoriasis and atopic dermatitis. Farah Sheikh reviews the specialized junctional components in cardiomyocytes of the cardiac conduction system and Robert Gourdie discusses how molecular complexes between sodium channels and gap junction proteins within the perijunctional microdomains within the intercalated disc facilitate conduction. Glenn Radice evaluates the role of N-cadherin in heart. Andre Kleber and Chris Chen explore new approaches to study junctional mechanotransduction in vitro with a focus on the effects of connexin ablation and the role of cadherins, respectively. To complement this series of reviews, we have interviewed Werner Franke, whose systematic documentation the tissue-specific complexity of desmosome composition and pioneering discovery of the cardiac area composita junction greatly facilitated elucidation of the role of desmosomal components in the pathophysiology of human heart disease.

OBJECTIVE: To determine the role of ANK/Myb-binding protein 1a (MYBBP1a) and sphingosine kinase 1 (SPHK1) interactions in catabolic events of articular chondrocytes. METHOD: ANK/MYBBP1a and SPHK1 interactions were identified using yeast two-hybrid screening and co-immunoprecipitation. To determine the role of these interactions in catabolic events of articular chondrocytes, ank/ank and wild type mouse chondrocytes transfected with full-length or mutant ank expression vectors or femoral heads were treated with interleukin-1beta (IL-1beta) in the absence or presence of SPHK inhibitor. Catabolic marker mRNA levels were analyzed by real time PCR; proteoglycan loss using safranin O staining and MMP-13 immunostaining were determined in femoral head explants; NF-kappaB activity was determined by transfecting chondrocytes with a NF-kappaB-specific luciferase reporter and analyzing nuclear translocation of p65 by immunoblotting; MYBBP1a nuclear or cytoplasmic amounts were determined by immunohistochemistry and immunoblotting. RESULTS: The ANK N-terminal region interacted with SPHK1, whereas a cytoplasmic C-terminal loop interacted with MYBBP1a. Lack of ANK/MYBBP1a and SPHK1 interactions in ank/ank chondrocytes resulted in increased MYBBP1a nuclear amounts and decreased SPHK1 activity, and consequently decreased NF-kappaB activity, catabolic marker mRNA levels, proteoglycan loss, and MMP-13 immunostaining in IL-1beta-treated articular chondrocytes or femoral heads. Transfection with full-length ank expression vector reduced nuclear MYBBP1a amounts and fully restored SPHK and NF-kappaB activities in IL-1beta-treated ank/ank chondrocytes, whereas transfection with P5L or F376del mutant ank reduced nuclear MYBBP1a or increased SPHK activity, respectively, and consequently either transfection only partially restored NF-kappaB activity. CONCLUSION: ANK/MYBBP1a and SPHK1 interactions stimulate catabolic events in IL-1beta-mediated cartilage degradation.

Rapid advancements in the field of stem cell biology have led to many current efforts to exploit stem cells as therapeutic agents in regenerative medicine. However, current ex vivo cell manipulations common to most regenerative approaches create a variety of technical and regulatory hurdles to their clinical translation, and even simpler approaches that use exogenous factors to differentiate tissue-resident stem cells carry significant off-target side effects. We show that non-ionizing, low-power laser (LPL) treatment can instead be used as a minimally invasive tool to activate an endogenous latent growth factor complex, transforming growth factor-beta1 (TGF-beta1), that subsequently differentiates host stem cells to promote tissue regeneration. LPL treatment induced reactive oxygen species (ROS) in a dose-dependent manner, which, in turn, activated latent TGF-beta1 (LTGF-beta1) via a specific methionine residue (at position 253 on LAP). Laser-activated TGF-beta1 was capable of differentiating human dental stem cells in vitro. Further, an in vivo pulp capping model in rat teeth demonstrated significant increase in dentin regeneration after LPL treatment. These in vivo effects were abrogated in TGF-beta receptor II (TGF-betaRII) conditional knockout (DSPP(Cre)TGF-betaRII(fl/fl)) mice or when wild-type mice were given a TGF-betaRI inhibitor. These findings indicate a pivotal role for TGF-beta in mediating LPL-induced dental tissue regeneration. More broadly, this work outlines a mechanistic basis for harnessing resident stem cells with a light-activated endogenous cue for clinical regenerative applications.