Faculty Bibliography

Alterations in circulating lipids and ectopic lipid deposition impact on the risk of developing cardiovascular and metabolic diseases. Lipoprotein lipase (LPL) hydrolyzes fatty acids (FAs) from triglyceride (TAG)-rich lipoproteins including very low density lipoproteins (VLDLs) and chylomicrons, and regulates their distribution to peripheral tissues. Angiopoietin-like 4 (ANGPTL4) mediates the inhibition of LPL activity under different circumstances. Accumulating evidence associates ANGPTL4 directly with the risk of atherosclerosis and type 2 diabetes (T2D). This review focuses on recent findings on the role of ANGPTL4 in metabolic and cardiovascular diseases. We highlight human and murine studies that explore ANGPTL4 functions in different tissues and how these effect disease development through possible autocrine and paracrine forms of regulation.

BACKGROUND:Fibroproliferative disorders result in excessive scar formation, are associated with high morbidity, and cost billions of dollars every year. Of these, keloid disease presents a particularly challenging clinical problem because the cutaneous scars progress beyond the original site of injury. Altered mechanotransduction has been implicated in keloid development, but the mechanisms governing scar progression into the surrounding tissue remain unknown. The role of mechanotransduction in keloids is further complicated by the differential mechanical properties of keloids and the surrounding skin. METHODS:The authors used human mechanical testing, finite element modeling, and immunohistologic analyses of human specimens to clarify the complex interplay of mechanical stress, strain, and stiffness in keloid scar progression. RESULTS:Changes in human position (i.e., standing, sitting, and supine) are correlated to dynamic changes in local stress/strain distribution, particularly in regions with a predilection for keloids. Keloids are composed of stiff tissue, which displays a fibrotic phenotype with relatively low proliferation. In contrast, the soft skin surrounding keloids is exposed to high mechanical strain that correlates with increased expression of the caveolin-1/rho signaling via rho kinase mechanotransduction pathway and elevated inflammation and proliferation, which may lead to keloid progression. CONCLUSIONS:The authors conclude that changes in human position are strongly correlated with mechanical loading of the predilection sites, which leads to increased mechanical strain in the peripheral tissue surrounding keloids. Furthermore, increased mechanical strain in the peripheral tissue, which is the site of keloid progression, was correlated with aberrant expression of caveolin-1/ROCK signaling pathway. These findings suggest a novel mechanism for keloid progression.

Melanocyte stem cells (McSCs) reside in the hair follicle bulge/secondary hair germ niche where they are essential for hair pigmentation and have the potential to also regulate epidermal pigmentation. A better understanding of the molecular mechanisms that govern these stem cells holds broad implications in pigmentation disorders including hair graying, vitiligo and melanoma. We show that Wnt signaling is temporarily activated in McSCs at the onset of hair follicle regeneration and is necessary and sufficient for their differentiation. We also show that endothelin receptor B signaling promotes proliferation and differentiation of McSCs, thereby dramatically enhances regeneration of hair and epidermal melanocytes. This effect however can only be seen in the presence of active Wnt signaling that is initiated by Wnt ligand secretion from epithelial niche. Upon skin injury or UVB irradiation, Wnt signaling and Edn signaling promote McSCs to regenerate epidermal melanocytes in the skin. Finally, we show that Wnt and Edn signals, secreted during melanocyte regeneration, can be hijacked to promote McSC malignant transformation during melanoma induction

We previously showed that KLF4, a gene highly expressed in murine prostate stem cells, blocks the progression of indolent intraepithelial prostatic lesions into aggressive and rapidly growing tumors. Here, we show that the anti-tumorigenic effect of KLF4 extends to PC3 human prostate cancer cells growing in the bone. We compared KLF4 null cells with cells transduced with a DOX-inducible KLF4 expression system, and find KLF4 function inhibits PC3 growth in monolayer and soft agar cultures. Furthermore, KLF4 null cells proliferate rapidly, forming large, invasive, and osteolytic tumors when injected into mouse femurs, whereas KLF4 re-expression immediately after their intra-femoral inoculation blocks tumor development and preserves a normal bone architecture. KLF4 re-expression in established KLF4 null bone tumors inhibits their osteolytic effects, preventing bone fractures and inducing an osteogenic response with new bone formation. In addition to these profound biological changes, KLF4 also induces a transcriptional shift from an osteolytic program in KLF4 null cells to an osteogenic program. Importantly, bioinformatic analysis shows that genes regulated by KLF4 overlap significantly with those expressed in metastatic prostate cancer patients and in three individual cohorts with bone metastases, strengthening the clinical relevance of the findings in our xenograft model.

Hyaluronan (HA) fragments have been proposed to elicit defensive or pro-inflammatory responses in many cell types. For articular chondrocytes in an inflammatory environment, studies have failed to reach consensus on the endogenous production or effects of added HA fragments. The present study was undertaken to resolve this discrepancy. Cultured primary human articular chondrocytes were exposed to the inflammatory cytokine IL-1β, and then tested for changes in HA content/size in conditioned medium, and for the expression of genes important in HA binding/signaling or metabolism, and in other catabolic/anabolic responses. Changes in gene expression caused by enzymatic degradation of endogenous HA, or addition of exogenous HA fragments, were examined. IL-1β increased the mRNA levels for HA synthases HAS2/HAS3 and for the HA-binding proteins CD44 and TSG-6. mRNA levels for TLR4 and RHAMM were very low and were little affected by IL-1β. mRNA levels for catabolic markers were increased, while type II collagen (α1(II)) and aggrecan were decreased. HA concentration in the conditioned medium was increased, but the HA was not degraded. Treatment with recombinant hyaluronidase or addition of low endotoxin HA fragments did not elicit pro-inflammatory responses. Our findings showed that HA fragments were not produced by IL-1β-stimulated human articular chondrocytes in the absence of other sources of reactive oxygen or nitrogen species, and that exogenous HA fragments from oligosaccharides up to about 40 kDa in molecular mass were not pro-inflammatory agents for human articular chondrocytes, probably due to low expression of TLR4 and RHAMM in these cells.

Cardiolipin[MS1]  (CL) is the signature phospholipid of mitochondrial membranes. Although it has long been known that CL plays an important role in mitochondrial bioenergetics, recent evidence in the yeast model indicates that CL is also essential for intermediary metabolism. To gain insight into the function of CL in energy metabolism in mammalian cells, here we analyzed the metabolic flux of [U-13C]glucose in a mouse C2C12 myoblast cell line, TAZ-KO, which is CL-deficient because of a CRISPR/Cas9-mediated knockout of the CL-remodeling enzyme tafazzin (TAZ). TAZ-KO cells exhibited decreased flux of [U-13C]glucose to [13C]acetyl-CoA and M2 and M4 isotopomers of TCA cycle intermediates. Activity of pyruvate carboxylase (PC), the predominant enzyme for anaplerotic replenishing of the TCA cycle, was elevated in the TAZ-KO cells, which also exhibited increased sensitivity to the PC inhibitor phenylacetate. We attributed a decreased carbon flux from glucose to acetyl-CoA in the TAZ-KO cells to a ~50% decrease in pyruvate dehydrogenase (PDH) activity, which was observed in both TAZ-KO cells and cardiac tissue from TAZ-KO mice. Protein-lipid overlay experiments revealed that PDH binds to CL, and supplementing digitonin-solubilized TAZ-KO mitochondria with CL restored PDH activity to wildtype levels. Mitochondria from TAZ-KO cells exhibited an increase in phosphorylated PDH, levels of which were reduced in the presence of supplemented CL. These findings indicate that CL is required for optimal PDH activation, generation of acetyl-CoA, and TCA cycle function, findings that link the key mitochondrial lipid CL to TCA cycle function and energy metabolism.

Interfollicular epidermal melanocytes are continually subjected to environmental challenges and activate protective stress responses for survival. Dysregulation of these responses may increase susceptibility to depigmentation typical of chemical leukoderma (depigmentation limited to site of exposure) and vitiligo (progressive depigmentation due to an autoimmune response). We delineated the response of melanocytes from normally pigmented individuals (NMs) to challenge with the chemotoxins monobenzone (MBEH) and 4-tertiary butyl phenol (4-TBP) and determined that the unfolded protein stress response (UPR) was activated following exposure. The UPR, a key survival pathway, is activated when the homeostasis of the Endoplasmic Reticulum (ER) is disrupted, for example by cellular oxidative stress induced by chemotoxin exposure. The UPR, which consists of three signal transduction pathways initiated by PERK, IRE1 or ATF6 respectively, promotes restoration of ER homeostasis and survival. In this study, we assessed the cytoprotective effect of the PERK arm of the UPR. PERK phosphorylates a number of proteins including the eukaryotic translation initiation factor, eIF2alpha. We treated melanocytes with the PERK kinase inhibitor GSK2606414 and confirmed efficacy by monitoring eIF2alpha phosphorylation, which was reduced after treatment. Melanocytes were then dosed with MBEH in the presence or absence of GSK2606414. The inhibitor sensitized melanocytes to MBEH (Cleaved/c-PARP observed with 250 muM MBEH + GSK2606414, compared to 400 muM MBEH + vehicle). To further investigate the role of PERK in melanocytes, we used a gene silencing (shRNA) approach to knockdown expression. An 88% decrease in viability (p < 0.0001) was observed 3 days post-infection (shPERK versus scrambled/shNT). Cultures were maintained for 14 days when viability was found to be improved (40% decrease in viability, p < 0.0001). Melanocytes that survived prolonged PERK downregulation adapted and could be maintained in culture (shPERKLT). Survival correlated with a paradoxical increase in phospho-eIF2alpha and reduced sensitivity to MBEH (c-PARP observed with 500 muM MBEH in shPERKLT versus 400 muM MBEH in shNT cells). Intriguingly, while eIF2alpha is not typically phosphorylated in unstressed cells, a fraction of eIF2alpha was phosphorylated in melanocytes at baseline. Thus PERK may play a role in determining melanocyte viability and sensitivity to chemotoxins

Vitiligo, an acquired depigmentation disorder, results from autoimmune targeting of melanocytes. Vitiligo can be triggered following exposure to phenols such as monobenzone (MBEH) and 4-tertiary butyl phenol (4-TBP). Melanocytes from individuals with idiopathic vitiligo (trigger is not known) are more sensitive to MBEH and 4-TBP. We hypothesized that stress response pathways activated following exposure to vitiligo triggers may be dysregulated in individuals who develop the disorder. We delineated the response of melanocytes from normally pigmented individuals (NMs) to challenge with MBEH and 4-TBP and identified two key survival pathways activated following exposure: the NRF2-regulated antioxidant response and the unfolded protein stress response (UPR). NRF2 knockdown sensitized NMs to MBEH (p < 0.0001), while NRF2 activation by knockdown of its repressor KEAP significantly decreased sensitivity (p < 0.0001). Similarly, inhibition of the PERK-eIF2alpha arm of the UPR with the chemical inhibitor GSK2606414 increased sensitivity to MBEH (cleaved PARP observed at 250 muM MBEH with GSK2606414 and 400 muM without). Activation of NRF2-regulated antioxidant responses and PERK-mediated phosphorylation of eIF2alpha following MBEH exposure was impaired in melanocytes from individuals who developed vitiligo. We have thus identified two stress response pathways that may be dysfunctional in vitiligo and contribute to the onset of depigmentation

BACKGROUND:Observational studies support an association between a low blood 25-hydroxyvitamin D level and the risk of type 2 diabetes. However, whether vitamin D supplementation lowers the risk of diabetes is unknown. METHODS:or placebo, regardless of the baseline serum 25-hydroxyvitamin D level. The primary outcome in this time-to-event analysis was new-onset diabetes, and the trial design was event-driven, with a target number of diabetes events of 508. RESULTS:A total of 2423 participants underwent randomization (1211 to the vitamin D group and 1212 to the placebo group). By month 24, the mean serum 25-hydroxyvitamin D level in the vitamin D group was 54.3 ng per milliliter (from 27.7 ng per milliliter at baseline), as compared with 28.8 ng per milliliter in the placebo group (from 28.2 ng per milliliter at baseline). After a median follow-up of 2.5 years, the primary outcome of diabetes occurred in 293 participants in the vitamin D group and 323 in the placebo group (9.39 and 10.66 events per 100 person-years, respectively). The hazard ratio for vitamin D as compared with placebo was 0.88 (0.95% confidence interval, 0.75 to 1.04; P = 0.12). The incidence of adverse events did not differ significantly between the two groups. CONCLUSIONS:supplementation at a dose of 4000 IU per day did not result in a significantly lower risk of diabetes than placebo. (Funded by the National Institute of Diabetes and Digestive and Kidney Diseases and others; D2d ClinicalTrials.gov number, NCT01942694.).

RIP1 regulates cell death and inflammation and is believed to play an important role in contributing to a variety of human pathologies, including immune-mediated inflammatory diseases and cancer. While small-molecule inhibitors of RIP1 kinase have been advanced to the clinic for inflammatory diseases and CNS indications, RIP1 inhibitors for oncology indications have yet to be described. Herein we report on the discovery and profile of GSK3145095 (compound 6). Compound 6 potently binds to RIP1 with exquisite kinase specificity and has excellent activity in blocking RIP1 kinase-dependent cellular responses. Highlighting its potential as a novel cancer therapy, the inhibitor was also able to promote a tumor suppressive T cell phenotype in pancreatic adenocarcinoma organ cultures. Compound 6 is currently in phase 1 clinical studies for pancreatic adenocarcinoma and other selected solid tumors.