Faculty Bibliography

Microsomal triglyceride transfer protein (MTP) is needed to assemble chylomicrons in the endoplasmic reticulum (ER) of enterocytes. We explored the role of an ER stress protein, inositol-requiring enzyme 1beta (IRE1beta), in regulating this process. High-cholesterol and high-fat diets decreased intestinal IRE1beta mRNA in wild-type mice. Ire1b(-/-) mice fed high-cholesterol and high-fat diets developed more pronounced hyperlipidemia because these mice secreted more chylomicrons and expressed more intestinal MTP, though not hepatic MTP, than wild-type mice did. Chylomicron secretion and MTP expression also were increased in primary enterocytes isolated from cholesterol-fed Ire1b(-/-) mice. There was no correlation between ER stress and MTP expression. Instead, cell culture studies revealed that IRE1beta, but not its ubiquitous homolog IRE1alpha, decreased MTP mRNA through increased posttranscriptional degradation. Conversely, knockdown of IRE1beta enhanced MTP expression. These studies show that IRE1beta plays a role in regulating MTP and in chylomicron production

Middle T antigen (MT) is the principal oncoprotein of murine polyomavirus. Experiments on the acute immediate effects of MT expression on cellular RNA levels showed that expression of osteopontin (OPN) was strongly induced by MT expression. Osteopontin is a protein known to be associated with cancer. It has a role in tumor progression and invasion. Protein analysis confirmed that MT induced the secretion of OPN into the extracellular medium. Expression of antisense OPN RNA had no effect on the growth of MT-transformed cells. However, it had a strong effect on the ability of MT transformants to migrate or to fill a wound. Analysis of MT mutants implicated both the SHC and phosphatidylinositol 3-kinase pathways in OPN induction. Reporter assays showed that MT regulated the OPN promoter through two of its PEA3 (polyoma enhancer activator 3) sites. As critical PEA3 sites are also part of the polyomavirus enhancer, the same signaling important for viral replication also contributes to virally induced metastatic potential.

In both normal development and in a variety of pathological conditions, epithelial cells can acquire migratory and invasive properties. Border cells in the Drosophila ovary provide a genetically tractable model for elucidating the mechanisms controlling such behaviors. Here we report the identification of a mutant, apontic (apt), in which the migratory population expanded and separation from the epithelium was impeded. This phenotype resembled gain-of-function of JAK/STAT activity. Gain-of-function of APT also mimicked loss of function of STAT and its key downstream target, SLBO. APT expression was induced by STAT, which bound directly to sites in the apt gene. The data suggest that a regulatory circuit between STAT, APT, and SLBO functions to convert an initially graded signal into an all-or-nothing activation of JAK/STAT and thus to proper cell specification and migration. These findings are supported by a mathematical model, which accurately simulates wild-type and mutant phenotypes.

Gene deletion experiments have shown that the three TGFbeta isoforms regulate distinct developmental processes. Recent work by our group and others showed that the integrins alphavbeta6 and alphavbeta8 activate latent forms of TGFbeta1 and TGFbeta3. This raises the possibility that TGFbeta1 and TGFbeta3 act redundantly in developmental processes where both isoforms are expressed and activation is by integrins. To investigate this issue, we generated mice with defective integrin-mediated TGFbeta1 activation (Tgfb1(RGE/RGE)) that were also homozygous for a null mutation in the TGFbeta3 gene. Tgfb1(RGE/RGE); Tgfb3(-/-) mice have severely perturbed development of the brain vasculature that is highly similar to that in mice lacking alphavbeta8. Some Tgfb1(RGE/RGE); Tgfb3(+/-) and Tgfb1(RGE/RGE); Tgfb3(+/+) mice have milder, background-dependent versions of the phenotype. In addition, we found that Tgfb3 gene status influences embryonic lethality due to TGFbeta1 deficiency after limited backcrossing to the BALB/c background. Conversely, Tgfb1 gene status modifies the extent of palate fusion in Tgfb3(-/-) mice after limited backcrossing to the ICR background. Our results are consistent with a functional connection between TGFbeta1 and TGFbeta3 during development based on a shared mechanism of activation

A screen of a library of 2,000 drugs and natural products in murine melanocytes identified 10 tricyclic antidepressants (TCAs) as compounds that potently decreased intracellular melanin content. The rank order of potency of these compounds for decreasing melanin content was different than their relative potencies as antidepressants. These compounds had no effect on either the level or the enzymatic activity of cellular tyrosinase (Tyr). Increased presence of both Tyr and melanin in the culture media was observed in treated melanocytes. Immunofluorescence localization revealed that these compounds decreased intracellular melanin content by disrupting the intracellular trafficking of Tyr gene family proteins. In treated melanocytes, Tyr, Tyr-related protein 1, and dopachrome tautomerase accumulated in enlarged granules distributed throughout the cytoplasm. Colocalization of Tyr with lysosome-associated membrane protein 1 was observed within many of these granules. Partial colocalization of Tyr with the Hermansky-Pudlak syndrome 1 gene product observed in control melanocytes was abolished by TCA treatment. Our results show that these compounds decreased intracellular melanin content by altering the trafficking of Tyr gene family proteins and inducing abnormal secretion of Tyr. Results from our screening have implications for the design of products for skin lightening and treatment of hyperpigmentation

Fibrates are a class of lipid-lowering medication primarily used as second-line agents behind statins. Acting via the peroxisome proliferators-activated receptor-alpha, their main lipoprotein effects are to lower serum triglyceride levels and to raise high-density lipoprotein-cholesterol, with modest effects on low-density lipoprotein-cholesterol. However, many clinical trials indicate that fibrates may have benefits beyond simply altering one's lipid profile. Several angiographic studies show retardation in the progression of atherosclerotic lesions in coronary vessels. Although clinical trials have failed to show a reduction in mortality with fibrates, several post hoc analyses indicate that there may be a mortality benefit in patients with features of the metabolic syndrome. Given that fibrates are often used as second-line agents, it is essential they are safe to be given in combination with other agents, particularly statins and ezetimibe. Although the side-effect profile of fibrates includes gastrointestinal symptoms, increased liver function tests, a reversible rise in creatinine and myositis, in general, fibrates seem to be safe to use in combination with other lipid lowering medications. Thus far, fibrates have not shown a mortality benefit in randomized clinical trials; as a result, they cannot be considered first-line medication for the primary or secondary prevention of coronary artery disease

Id proteins play important roles in osteogenic differentiation; however, the molecular mechanism remains unknown. In this study, we established that inhibitor of differentiation (Id) proteins, including Id1, Id2, and Id3, associate with core binding factor alpha-1 (Cbfa1) to cause diminished transcription of the alkaline phosphatase (ALP) and osteocalcin (OCL) gene, leading to less ALP activity and osteocalcin (OCL) production. Id acts by inhibiting the sequence-specific binding of Cbfa1 to DNA and by decreasing the expression of Cbfa1 in cells undergoing osteogenic differentiation. p204, an interferon-inducible protein that interacts with both Cbfa1 and Id2, overcame the Id2-mediated inhibition of Cbfa1-induced ALP activity and OCL production. We show that 1) p204 disturbed the binding of Id2 to Cbfa1 and enabled Cbfa1 to bind to the promoters of its target genes and 2) that p204 promoted the translocation from nucleus to the cytoplasm and accelerated the degradation of Id2 by ubiquitin-proteasome pathway during osteogenesis. Nucleus export signal (NES) of p204 is required for the p204-enhanced cytoplasmic translocation and degradation of Id2, because a p204 mutant lacking NES lost these activities. Together, Cbfa1, p204, and Id proteins form a regulatory circuit and act in concert to regulate osteoblast differentiation

BACKGROUND: Clinical use of cancer gene therapy has been prevented by the inability to deliver high levels of local transgene expression with acceptable host toxicity. The authors' laboratory has developed an ex vivo technique to genetically modify free flaps to deliver immunotherapy locally without systemic toxicity. METHODS: Superficial inferior epigastric flaps were dissected in Fischer rats, perfused with a viral vector expressing the antitumor interleukin-12 (IL-12) for 1 hour, and re-anastomosed. Beneath the flaps was a bolus of 1 x 10(6) beta-human chorionic gonadotropin-secreting MADB-106 tumor cells. Tumor growth was monitored using beta-human chorionic gonadotropin levels (secreted by the tumor) and size. IL-12 expression in tissue was assessed by enzyme-linked immunosorbent assay. Tumor inflammatory infiltrate was assessed using immunohistologic staining (CD8 and CD161) and enzyme-linked immunosorbent assay (interferon-gamma). Serum levels of liver enzymes and histologic analysis were used to assess systemic toxicity. RESULTS: IL-12 expression was confirmed in the flap and surrounding tissue. The rate of tumor growth in the IL-12-treated group was significantly suppressed compared with the control group (p < 0.001). Liver enzyme levels remained normal, and histological evaluation of the liver, lung, and spleen revealed no evidence of inflammation in the treated group. CONCLUSIONS: Using genetically modified free flaps, the authors were able to deliver IL-12 directly into the local environment of a tumor and suppress its growth without eliciting toxic systemic effects. This technique could provide valuable adjuvant treatment after oncologic surgery for soft-tissue cancers, with the transduced flap reconstructing the defect and supplying a therapeutic agent to the resected tumor bed.

PURPOSE: Previous study has shown that the absence of uroplakin II can cause urinary tract dysfunction, including vesicoureteral reflux and renal abnormalities, as well as micturition pattern changes. We developed a simple surrogate measure of bladder function using ultraviolet visualization of urinary voiding patterns in a uroplakin II knockout mouse animal model. MATERIALS AND METHODS: Three male and 3 female WT mice, and 3 male and 3 female uroplakin II knockout mice were evaluated by cystometric analysis and voiding pattern markings. Voiding pattern markings were graded by independent observers on a scale of 1 to 5 according to the degree of dispersion of voided urine. Statistical analysis was then used to correlate voiding dispersion grades with cystometric parameters in the same mice. RESULTS: The degree of dispersion of voiding pattern markings correlated with several measures of bladder function. Specifically the Pearson correlation coefficients for the observed voiding patterns highly correlated with baseline pressure, threshold pressure and intermicturition pressure measurements made during conscious cystometry in these mice (p <0.05). CONCLUSIONS: Ultraviolet visualization of urinary voiding patterns of mice correlated well with certain measures of standard cystometric evaluations. As such, this method provides a simple, noninvasive method of evaluating mouse bladder function. Implementation of this methodology, which can potentially be automated for high throughput analysis, can accelerate the development of novel therapy for certain important aspects of bladder disease/dysfunction

PURPOSE: Beta-adrenergic receptor (AR) antagonists are frequently prescribed ophthalmic drugs, yet previous investigations into how catecholamines affect corneal wound healing have yielded conflicting RESULTS: With the use of an integrated pharmacologic and genetic approach, the authors investigated how the beta-AR impacts corneal epithelial healing. METHODS: Migratory rates of cultured adult murine corneal epithelial (AMCE) cells and in vivo corneal wound healing were examined in beta2-AR(+/+) and beta2-AR(-/-) mice. Signaling pathways were evaluated by immunoblotting. results. The beta-AR agonist isoproterenol decreased AMCE cell migratory speed to 70% of untreated controls, and this was correlated with a 0.60-fold decrease in levels of activated phospho-ERK (P-ERK). Treatment with the beta-AR antagonist (timolol) increased speed 33% and increased P-ERK 2.4-fold (P < 0.05). The same treatment protocols had no effect on AMCE cells derived from beta2-AR(-/-) mice; all treatment groups showed statistically equivalent migratory speeds and ERK phosphorylation. In beta2-AR(+/+) animals, the beta-AR agonist (isoproterenol) delayed the rate of in vivo corneal wound healing by 79%, whereas beta-AR antagonist (timolol) treatment increased the rate of healing by 16% (P < 0.05) compared with saline-treated controls. In contrast, in the beta2-AR(-/-) mice, all treatment groups demonstrated equivalent rates of wound healing. Additionally, murine corneal epithelial cell expressed the catecholamine-synthesizing enzyme tyrosine hydroxylase and detectable levels of epinephrine (184.5 pg/mg protein). CONCLUSIONS: The authors provide evidence of an endogenous autocrine catecholamine signaling pathway dependent on an intact beta2-AR for the modulation of corneal epithelial wound repair