Faculty Bibliography

BRCA1 germline mutations confer a substantial risk of breast cancer, attributed to a trifecta of defective DNA repair, genomic instability, and abnormal mammary lineage commitment.We developed a novel mouse model of Brca1 deficiency, characterized by an extremely abnormal mammary phenotype. At 28 days of age, affected animals had more numerous and larger terminal end buds, in which cell proliferation rate was significantly higher compared to wildtype mice (p<0.01). As animals aged, their mammary glands exhibited ductal dilatation, cystic changes, pre-malignant hyperplastic lesions, and ductal carcinoma in situ. An index of genomic instability based on in situ analysis of centrosomes was significantly increased in Brca1-deficient epithelium compared to wildtype tissue (p<0.01). We identified a population of mammary basal epithelial cells expressing both luminal and myoepithelial lineage markers that represented 22.5% of the basal cell population in Brca1-deficient mice, compared to 3.9% in wildtypes (p<0.01).Phosphorylation of IGF-I receptor (IGF-IR) and its downstream signaling mediators were increased in affected mice. To determine if IGF-I inhibition would reduce or reverse mammary gland abnormalities, we tested the effects of pasireotide, a somatostatin analog that inhibits IGF-I action by a direct effect in the mammary gland and also by reducing serum GH. In 4-month-old mice, 7 days of treatment with pasireotide (vs. vehicle) caused a reduction in primary, secondary, and tertiary duct width (for primary ducts: 109.2 mum with pasireotide vs. 313.8 mum with vehicle, p<0.01), halved the epithelial cell proliferation rate (10.6% vs. 20.8%, p<0.05), normalized the centrosome aberration index (1.1 vs. 2.3, p=0.01), and eliminated the aberrant epithelial population (4.8% vs. 22.5%, p<0.01). Phosphorylation of IGF-IR and its downstream mediators was inhibited. Results were similar after treatment for 7 days with PQ401, a small molecule inhibitor of IGF-IR, substantiating the IGF-I dependence of the Brca1-deficient phenotype response to pasireotide.In conclusion, we present a novel mouse model of Brca1 deficiency with pre-malignant and early malignant changes, which is extremely sensitive to short-term IGF-I inhibition by pasireotide. Translation of these findings may prove effective in preventing breast cancer in women with BRCA1 mutations

Granulin-epithelin precursor (GEP) is an autocrine growth factor that has been implicated in embryonic development, tissue repair, tumorigenesis, and inflammation. Here we report that GEP was expressed in skeletal muscle tissue and its level was differentially altered in the course of C2C12 myoblast fusion. The GEP expression during myoblast fusion was a consequence of MyoD transcription factor binding to several E-box (CANNTG) sequences in the 5'-flanking regulatory region of GEP gene, followed by transcription. Recombinant GEP potently inhibited myotube formation from C2C12 myoblasts whereas the knockdown of endogenous of GEP via a siRNA approach accelerated the fusion of myoblasts to myotubes. Interestingly, the muscle fibers of GEP knockdown mice were larger in number but noticeably smaller in size when compared to the wild-type. Mechanistic studies revealed that during myoblast fusion, the addition of GEP led to remarkable reductions in the expressions of muscle-specific transcription factors, including MyoD. In addition, the regulation of myotube formation by GEP is mediated by the anti-myogenic factor JunB, which is upregulated following GEP stimulation. Thus, GEP growth factor, JunB, and MyoD transcription factor form a regulatory loop and act in concert in the course of myogenesis

BACKGROUND: Nasal polyposis (NP) is a Th2-skewed inflammatory disorder, but it is unclear what role regulatory T cells (T-reg) play in disease pathology. We investigated the expression profiles of T-reg and T-helper-cell-associated genes and their response to glucocorticosteroid (GC) treatment in Chinese patients with NP. METHODS: Biopsies were obtained from 29 non-treated NP patients for comparison with inferior turbinates collected from healthy controls. In 13 patients, NP samples were collected both before and after short-term oral GC treatment. Levels of mRNA for T-cell markers were determined by microarray and quantitative PCR. Cellular infiltrates were assessed by histo- and immunohistochemistry. RESULTS: FOXP3(+) T-reg were increased in GC-naive NP, and numbers were negatively correlated with eosinophil infiltration. Helios staining was not detected, suggesting that FOXP3(+) cells in NP are not thymus-derived T-reg. Compared with controls, mRNA levels corresponding to T-reg genes were significantly increased in NP (FOXP3, TGFB1, IL10, SMAD3, IL2RA, and JAK3), but transcription factors associated with Th2 (GATA3) or Th17 responses (RORc) were significantly reduced. FOXP3 mRNA levels positively correlated with other T-reg cell markers. Microarray analysis showed that most Th2-related markers (e.g., Eotaxin-1, CCL13, and CCL18) were upregulated in GC-naive NP vs controls. GC therapy significantly suppressed eosinophilic inflammation in NP, but did not significantly alter the expression levels of T-reg/Th2-associated genes. CONCLUSIONS: Upregulation of FOXP3(+) -inducible T-reg cells and downregulation of Th2 and Th17 markers in NP indicate a regulatory response occurring at a site of persistent mucosal inflammation. However, immune regulation fails to control the underlying tissue pathology. Expression of T-reg/Th2 markers after GC treatment was unaltered, suggesting that T-cell-driving NP inflammatory mediators are GC resistant.

We report that neuronal overexpression of the endogenous inhibitor of calpains, calpastatin (CAST), in a mouse model of human Alzheimer's disease (AD) beta-amyloidosis, the APP23 mouse, reduces beta-amyloid (Abeta) pathology and Abeta levels when comparing aged, double transgenic (tg) APP23/CAST with APP23 mice. Concurrent with Abeta plaque deposition, aged APP23/CAST mice show a decrease in the steady-state brain levels of the amyloid precursor protein (APP) and APP C-terminal fragments (CTFs) when compared with APP23 mice. This CAST-dependent decrease in APP metabolite levels was not observed in single tg CAST mice expressing endogenous APP or in younger, Abeta plaque predepositing APP23/CAST mice. We also determined that the CAST-mediated inhibition of calpain activity in the brain is greater in the CAST mice with Abeta pathology than in non-APP tg mice, as demonstrated by a decrease in calpain-mediated cytoskeleton protein cleavage. Moreover, aged APP23/CAST mice have reduced extracellular signal-regulated kinase 1/2 (ERK1/2) activity and tau phosphorylation when compared with APP23 mice. In summary, in vivo calpain inhibition mediated by CAST transgene expression reduces Abeta pathology in APP23 mice, with our findings further suggesting that APP metabolism is modified by CAST overexpression as the mice develop Abeta pathology. Our results indicate that the calpain system in neurons is more responsive to CAST inhibition under conditions of Abeta pathology, suggesting that in the disease state neurons may be more sensitive to the therapeutic use of calpain inhibitors.

Trans-epithelial migration describes the ability of migrating cells to cross epithelial tissues and occurs during development, infection, inflammation, immune surveillance, wound healing and cancer metastasis. Here we investigate Drosophila primordial germ cells (PGCs), which migrate through the endodermal epithelium. Through live imaging and genetic experimentation we demonstrate that PGCs take advantage of endodermal tissue remodeling to gain access to the gonadal mesoderm and are unable to migrate through intact epithelial tissues. These results are in contrast to the behavior of leukocytes, which actively loosen epithelial junctions to migrate, and raise the possibility that in other contexts in which migrating cells appear to breach tissue barriers, they are actually exploiting existing tissue permeability. Therefore, the use of active invasive programs is not the sole mechanism to infiltrate tissues.

Receptor activator of NF-B ligand (RANKL) stimulation leads to the activation of mitogen-activated protein kinase (MAPK)/AP-1 and Ca ²⁺-nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) signaling pathways in osteoclastogenesis. Targeting these pathways has been an encouraging strategy for bone-related diseases, such as postmenopausal osteoporosis. In this study, we examined the effects of caffeic acid 3,4-dihydroxy-phenethyl ester (CADPE) on osteoclastogenesis. In mouse bone marrow monocytes (BMMs) and RAW264.7 cells, CADPE suppressed RANKL-induced osteoclast differentiation and actin-ring formation in a dose-dependent manner within non-growth inhibitory concentrations at the early stage, while CADPE had no effect on macrophage colony-stimulating factor (M-CSF)-induced proliferation and differentiation. At the molecular level, CADPE inhibited RANKL-induced phosphorylation of MAPKs, including extracellular signal-regulated kinases 1/2 (ERK1/2), p38, and c-Jun N-terminal kinase (JNK), without significantly affecting the NF-B signaling pathway. CADPE abrogated RANKL-induced activator protein 1 (AP-1)/FBJ murine osteosarcoma viral oncogene homolog (c-Fos) nuclear translocation and activation. Overexpression of c-Fos prevented the inhibition by CADPE of osteoclast differentiation. Furthermore, CADPE suppressed RANKL-induced the tumor necrosis factor receptor associated factor 6 (TRAF6) interaction with c-src tyrosine kinase (c-Src), blocked RANKL-induced the phosphorylation of protein kinase B (AKT), and inhibited RANKL-induced Ca²⁺ oscillation. As a result, CADPE decreased osteoclastogenesis- related marker gene expression, including NFATc1, TRAP, cathepsin K, and c-Src. To test the effects of CADPE on osteoclast activity in vivo, we showed that CADPE prevented ovariectomy-induced bone loss by inhibiting osteoclast activity. Together, our data demonstrate that CADPE suppresses osteoclastogenesis and bone loss through inhibiting RANKL-induced MAPKs and Ca²⁺-NFATc1 signaling pathways. CADPE is a novel agent in the treatment of osteoclast-related diseases, such as osteoporosis

ATP-binding cassette transporter A1 (ABCA1) is a cholesterol transporter that transfers excess cellular cholesterol onto lipid-poor apolipoproteins. Given its critical role in cholesterol homeostasis, ABCA1 has been studied as a therapeutic target for Alzheimer's disease. Transcriptional regulation of ABCA1 by liver X receptor has been well characterized. However, whether ABCA1 expression is regulated at the posttranscriptional level is largely unknown. Identification of a novel pathway that regulates ABCA1 expression may provide new strategy for regulating cholesterol metabolism and amyloid beta (Abeta) levels. Since ABCA1 has an unusually long 3' untranslated region, we investigated whether microRNAs could regulate ABCA1 expression. We identified miR-106b as a novel regulator of ABCA1 expression and Abeta metabolism. miR-106b significantly decreased ABCA1 levels and impaired cellular cholesterol efflux in neuronal cells. Furthermore, miR-106b dramatically increased levels of secreted Abeta by increasing Abeta production and preventing Abeta clearance. Alterations in Abeta production and clearance were rescued by expression of miR-106b-resistant ABCA1. Taken together, our data suggest that miR-106b affects Abeta metabolism by suppressing ABCA1 expression.

Tremendous global efforts have been made to collect data on the HIV/AIDS epidemic. Yet, significant challenges remain for generating and analysing evidence to allocate resources efficiently and implement an effective AIDS response. India offers important lessons and a model for intelligent and integrated use of data on HIV/AIDS for an evidence-based response. Over the past 15 years, the number of data sources has expanded and the geographical unit of data generation, analysis and use for planning has shifted from the national to the state, district and now subdistrict level. The authors describe and critically analyse the evolution of data sets in India and how they have been utilised to better understand the epidemic, advance policy, and plan and implement an increasingly effective, well-targeted and decentralised national response to HIV and AIDS. The authors argue that India is an example of how 'know your epidemic, know your response' message can effectively be implemented at scale and presents important lessons to help other countries design their evidence generation systems.

OBJECTIVES: The reported long-term safety of kidney donation is inconsistent with the impairment of kidney function observed following nephrectomy for renal cell cancer. We aimed to investigate if indication for nephrectomy (kidney cancer vs. living donation) was an independent risk factor for kidney function deterioration. MATERIALS AND METHODS: Between 1985 and 2008, 124 patients with localized renal cell carcinoma who meet the criteria used for living donation, underwent radical nephrectomy (group 1) at our institution. Group 1 was retrospectively compared with 124 consecutive living donor nephrectomies (group 2) performed from 2004 to 2008. Kidney function evaluation was performed preoperatively and at 1, 2, 3, and 4 years postoperatively with calculation of estimated glomerular filtration rate through the Modification of Diet in Renal Disease (MDRD-eGFR) and the adjusted Cockroft and Gault (CG-eGFR) formula. Multivariate logistic regression included patients' characteristics and indication for nephrectomy as predictors of kidney function deterioration. RESULTS: Mean decrease in MDRD-eGFR was 30.4% and 32.4% in groups 1 and 2 (P = 0.30). Prevalence of chronic kidney disease (CKD), defined by MDRD-eGFR < 60 mL/min/m(2), varied from 42.3% to 71% in group 1 and from 41.6% to 56% in group 2 at different time points (P = 0.073). Prevalence of CKD at 4 years defined by MDRD-eGFR < 45 mL/min/m(2) was significantly increased in group 1 compared with group 2 (16.2% and 5.3%, P < 0.005, respectively). Linear regression analysis showed only baseline kidney function and patient age predicted a significant decrease in postoperative kidney function (P < 0.001 and P = 0.04). CONCLUSIONS: Renal cell carcinoma is not an independent risk factor for kidney function impairment following nephrectomy. Selected kidney cancer patients with few morbidities face the same deterioration of meanly 30% of kidney function compared with living donors, but their lower baseline function results in an increased risk for CKD.