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Figure S3 shows the effect of UCHL1 inhibitor, LDN57444 in vivo and in vitro.

Increasing evidence suggests an important role for deubiquitinating enzymes (DUBs) in modulating a variety of biological functions and diseases. We previously identified the upregulation of the DUB ubiquitin carboxyl terminal hydrolase 1 (UCHL1) in murine ventilator-induced lung injury (VILI). However, the role of UCHL1 in modulating vascular permeability, a cardinal feature of acute lung injury (ALI) in general, remains unclear. We investigated the role of UCHL1 in pulmonary endothelial cell (EC) barrier function in vitro and in vivo and examined the effects of UCHL1 on VE-cadherin and claudin-5 regulation, important adherens and tight junctional components, respectively. Measurements of transendothelial electrical resistance confirmed decreased barrier enhancement induced by hepatocyte growth factor (HGF) and increased thrombin-induced permeability in both UCHL1-silenced ECs and in ECs pretreated with LDN-57444 (LDN), a pharmacological UCHL1 inhibitor. In addition, UCHL1 knockdown (siRNA) was associated with decreased expression of VE-cadherin and claudin-5, whereas silencing of the transcription factor FoxO1 restored claudin-5 levels. Finally, UCHL1 inhibition in vivo via LDN was associated with increased VILI in a murine model. These findings support a prominent functional role of UCHL1 in regulating lung vascular permeability via alterations in adherens and tight junctions and implicate UCHL1 as an important mediator of ALI.

High-grade serous ovarian cancer (HGSOC) is characterized by chromosomal instability, DNA damage, oxidative stress, and high metabolic demand that exacerbate misfolded, unfolded, and damaged protein burden resulting in increased proteotoxicity. However, the underlying mechanisms that maintain protein homeostasis to promote HGSOC growth remain poorly understood. This study reports that the neuronal deubiquitinating enzyme, ubiquitin carboxyl-terminal hydrolase L1 (UCHL1), is overexpressed in HGSOC and maintains protein homeostasis. UCHL1 expression was markedly increased in HGSOC patient tumors and serous tubal intraepithelial carcinoma (HGSOC precursor lesions). High UCHL1 levels correlated with higher tumor grade and poor patient survival. UCHL1 inhibition reduced HGSOC cell proliferation and invasion, as well as significantly decreased the in vivo metastatic growth of ovarian cancer xenografts. Transcriptional profiling of UCHL1-silenced HGSOC cells revealed downregulation of genes implicated with proteasome activity along with upregulation of endoplasmic reticulum stress–induced genes. Reduced expression of proteasome subunit alpha 7 (PSMA7) and acylaminoacyl peptide hydrolase (APEH), upon silencing of UCHL1, resulted in a significant decrease in proteasome activity, impaired protein degradation, and abrogated HGSOC growth. Furthermore, the accumulation of polyubiquitinated proteins in the UCHL1-silenced cells led to attenuation of mTORC1 activity and protein synthesis, and induction of terminal unfolded protein response. Collectively, these results indicate that UCHL1 promotes HGSOC growth by mediating protein homeostasis through the PSMA7–APEH–proteasome axis. Implications: This study identifies the novel links in the proteostasis network to target protein homeostasis in HGSOC and recognizes the potential of inhibiting UCHL1 and APEH to sensitize cancer cells to proteotoxic stress in solid tumors.

High-grade serous ovarian cancer (HGSOC) is characterized by chromosomal instability, DNA damage, oxidative stress, and high metabolic demand, which exacerbate misfolded, unfolded and damaged protein burden resulting in increased proteotoxicity. However, the underlying mechanisms that maintain protein homeostasis to promote HGSOC growth remain poorly understood. In this study, we report that the neuronal deubiquitinating enzyme, ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) is overexpressed in HGSOC and maintains protein homeostasis. UCHL1 expression was markedly increased in HGSOC patient tumors and serous tubal intraepithelial carcinoma (HGSOC precursor lesions). High UCHL1 levels correlated with higher tumor grade and poor patient survival. UCHL1 inhibition reduced HGSOC cell proliferation and invasion through the outer layers of omentum as well as significantly decreased the in vivo metastatic tumor growth in ovarian cancer xenografts. Transcriptional profiling of UCHL1 silenced HGSOC cells revealed the down-regulation of genes implicated with proteasome activity along with the upregulation of endoplasmic reticulum (ER) stress-induced genes. Reduced expression of proteasome subunit alpha 7 (PSMA7) and acylaminoacyl peptide hydrolase (APEH) resulted in a significant decrease in proteasome activity, impaired protein degradation, and abrogated HGSOC growth. Furthermore, the accumulation of polyubiquitinated proteins in the UCHL1 silenced cells led to attenuation of mTORC1 activity and protein synthesis, and induction of terminal unfolded protein response. Collectively, these results indicate that UCHL1 promotes HGSOC growth by mediating protein homeostasis through the PSMA7-APEH-proteasome axis.Abstract FigureImplicationsThis study identifies the novel links in the proteostasis network to target protein homeostasis in HGSOC. It recognizes the potential of inhibiting UCHL1 and APEH to sensitize cancer cells to proteotoxic stress and as novel alternative therapeutic approaches.

BACKGROUND Cachexia frequently occurs in women with advanced ovarian cancer (OC), along with enhanced inflammation. Despite being responsible for one third of all cancer deaths, cachexia is generally under-studied in OC due to a limited number of pre-clinical animal models. We aimed to address this gap by characterizing the cachectic phenotype in a mouse model of OC. METHODS Nod SCID gamma mice (n = 6-10) were injected intraperitoneally with 1 × 107 ES-2 human OC cells to mimic disseminated abdominal disease. Muscle size and strength, as well as bone morphometry, were assessed. Tumour-derived effects on muscle fibres were investigated in C2C12 myotube cultures. IL-6 levels were detected in serum and ascites from tumour hosts, as well as in tumour sections. RESULTS In about 2 weeks, ES-2 cells developed abdominal tumours infiltrating omentum, mesentery, and adjacent organs. The ES-2 tumours caused severe cachexia with marked loss of body weight (-12%, P < 0.01) and ascites accumulation in the peritoneal cavity (4.7 ± 1.5 mL). Skeletal muscles appeared markedly smaller in the tumour-bearing mice (approximately -35%, P < 0.001). Muscle loss was accompanied by fibre atrophy, consistent with reduced muscle cross-sectional area (-34%, P < 0.01) and muscle weakness (-50%, P < 0.001). Body composition assessment by dual-energy X-ray absorptiometry revealed decreased bone mineral density (-8%, P < 0.01) and bone mineral content (-19%, P < 0.01), also consistent with reduced trabecular bone in both femurs and vertebrae, as suggested by micro-CT imaging of bone morphometry. In the ES-2 mouse model, cachexia was also associated with high tumour-derived IL-6 levels in plasma and ascites (26.3 and 279.6 pg/mL, respectively) and with elevated phospho-STAT3 (+274%, P < 0.001), reduced phospho-AKT (-44%, P < 0.001) and decreased mitochondrial proteins, as well as with increased protein ubiquitination (+42%, P < 0.001) and expression of ubiquitin ligases in the skeletal muscle of tumour hosts. Similarly, ES-2 conditioned medium directly induced fibre atrophy in C2C12 mouse myotubes (-16%, P < 0.001), consistent with elevated phospho-STAT3 (+1.4-fold, P < 0.001) and altered mitochondrial homoeostasis and metabolism, while inhibition of the IL-6/STAT3 signalling by means of INCB018424 was sufficient to restore the myotubes size. CONCLUSIONS Our results suggest that the development of ES-2 OC promotes muscle atrophy in both in vivo and in vitro conditions, accompanied by loss of bone mass, enhanced muscle protein catabolism, abnormal mitochondrial homoeostasis, and elevated IL-6 levels. Therefore, this represents an appropriate model for the study of OC cachexia. Our model will aid in identifying molecular mediators that could be effectively targeted in order to improve muscle wasting associated with OC.

Background: Ovarian cancer is the most lethal gynecologic malignancy. There is a lack of comprehensive investigation of disease initiation and progression, including gene expression changes during early metastatic colonization. Methods: RNA-sequencing (RNA-seq) was done with matched primary tumors and fallopian tubes (n = 8 pairs) as well as matched metastatic and primary tumors (n = 11 pairs) from ovarian cancer patients. Since these are end point analyses, it was combined with RNA-seq using high-grade serous ovarian cancer cells seeded on an organotypic three-dimensional (3D) culture model of the omentum, mimicking early metastasis. This comprehensive approach revealed key changes in gene expression occurring in ovarian cancer initiation and metastasis, including early metastatic colonization. Results: 2987 genes were significantly deregulated in primary tumors compared to fallopian tubes, 845 genes were differentially expressed in metastasis compared to primary tumors and 304 genes were common to both. An assessment of patient metastasis and 3D omental culture model of early metastatic colonization revealed 144 common genes that were altered during early colonization and remain deregulated even in the fully developed metastasis. Deregulation of the matrisome was a key process in early and late metastasis. Conclusion: These findings will help in understanding the key pathways involved in ovarian cancer progression and eventually targeting those pathways for therapeutic interventions.

Aim Fracture risk assessment tool® calculations can be performed with or without addition of bone mineral density; however, the impact of this addition on fracture risk assessment tool® scores has not been studied in Indian women. Given the limited availability and high cost of bone mineral density testing in India, it is important to know the influence of bone mineral density on fracture risk assessment tool® scores in Indian women. Therefore, our aim was to assess the contribution of bone mineral density in fracture risk assessment tool® outcome in Indian women. Methods Apparently healthy postmenopausal Indian women (n = 506), aged 40–72 years, without clinical risk factors for bone disease, were retrospectively selected, and their fracture risk assessment tool® scores calculated with and without bone mineral density were compared. Results Based on WHO criteria, 30% women were osteoporotic, 42.9% were osteopenic and 27.1% had normal bone mineral density. Fracture risk assessment tool® scores for risk of both major osteoporotic fracture and hip fracture significantly increased on including bone mineral density ( P 0.05, whereas, for hip fracture risk number of women eligible without bone mineral density was 2 and with bone mineral density was 17, P Conclusion Until the establishment of country-specific medication intervention thresholds, bone mineral density should be included while calculating fracture risk assessment tool® scores in Indian women.

We previously reported protective effects of GADD45a (growth arrest and DNA damage-inducible gene 45 alpha) in murine ventilator-induced lung injury (VILI) via effects on Akt-mediated endothelial cell signaling. In the present study we investigated the role of GADD45a in separate murine models of radiation- and bleomycin-induced lung injury. Initial studies of wild-type mice subjected to single-dose thoracic radiation (10 Gy) confirmed a significant increase in lung GADD45a expression within 24 h and persistent at 6 wk. Mice deficient in GADD45a (GADD45a−/−) demonstrated increased susceptibility to radiation-induced lung injury (RILI, 10 Gy) evidenced by increased bronchoalveolar lavage (BAL) fluid total cell counts, protein and albumin levels, and levels of inflammatory cytokines compared with RILI-challenged wild-type animals at 2 and 4 wk. Furthermore, GADD45a−/−mice had decreased total and phosphorylated lung Akt levels both at baseline and 6 wk after RILI challenge relative to wild-type mice while increased RILI susceptibility was observed in both Akt+/−mice and mice treated with an Akt inhibitor beginning 1 wk prior to irradiation. Additionally, overexpression of a constitutively active Akt1 transgene reversed RILI-susceptibility in GADD45a−/−mice. In separate studies, lung fibrotic changes 2 wk after treatment with bleomycin (0.25 U/kg IT) was significantly increased in GADD45a−/−mice compared with wild-type mice assessed by lung collagen content and histology. These data implicate GADD45a as an important modulator of lung inflammatory responses across different injury models and highlight GADD45a-mediated signaling as a novel target in inflammatory lung injury clinically.

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High-grade serous ovarian cancer (HGSOC) is the most lethal subtype of ovarian cancer with high case-to-fatality ratio. However, not much advancement has been seen in the field in terms of novel adjuvant therapies. Somatic or germline mutations in HGSOC along with copy number variations results in intratumoral genetic heterogeneity, which leads to tumor growth and development of chemoresistant clones and treatment resistance. Moreover, molecular heterogeneity also leads to the accumulation of aberrant peptides and protein complexes with altered stoichiometry, causing extensive proteotoxic stress in cancer cells and adding to the known misfolded proteins stress in cancer cells due to enhanced protein synthesis, thus making these cancer cells more dependent on the protein clearance mechanisms. Not much is known about the molecular targets from the proteostasis network and their therapeutic potential in HGSOC. Here we explore the regulation and role of different components of ubiquitin proteasome system (UPS) in HGSOC and investigate the therapeutic effect of small-molecule inhibitors of UPS. We identified overexpression of deubiquitinating enzyme, ubiquitin carboxyl-terminal hydrolase 1 (UCHL1) in HGSOC, and high UCHL1 levels were correlated with the disease stage, tumor grade, and poor clinical outcome. UCHL1 overexpression in HGSOC was driven by mutant p53-mediated H3K4 trimethylation at the UCHL1 regulatory region. Mutant p53-UCHL1 axis transcriptionally promoted the expression of proteasomal subunit alpha 7 (PSMA7) and acylaminoacyl-peptide hydrolase (APEH), resulting in increased proteasome activity in HGSOC. Silencing UCHL1 or inhibiting proteasome activity in vitro and in vivo remarkably reduced HGSOC growth. Together, these results highlight the novel mechanisms of regulation of proteostasis in HGSOC and potential therapies targeting regulators of proteasome pathway. Citation Format: Kinzie Lighty, Yonghyun Shin, Fahmi Mesmer, Harikrishna Nakshatri, Marcin Iwanicki, Sumegha Mitra. Targeting new links in the proteostasis network as novel therapies in high-grade serous ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A54.

In high-grade serous ovarian cancer (OC), chemotherapy eliminates the majority of tumor cells, leaving behind residual tumors enriched in OC stem cells (OCSC). OCSC, defined as aldehyde dehydrogenase-positive (ALDH+), persist and contribute to tumor relapse. Inflammatory cytokine IL-6 is elevated in residual tumors after platinum treatment, and we hypothesized that IL-6 plays a critical role in platinum-induced OCSC enrichment. We demonstrate that IL-6 regulates stemness features of OCSC driven by ALDH1A1 expression and activity. We show that platinum induces IL-6 secretion by cancer-associated fibroblasts in the tumor microenvironment, promoting OCSC enrichment in residual tumors after chemotherapy. By activating STAT3 and upregulating ALDH1A1 expression, IL-6 treatment converted non-OCSC to OCSC. Having previously shown altered DNA methylation in OCSC, we show here that IL-6 induces DNA methyltransferase 1 (DNMT1) expression and the hypomethylating agent (HMA) guadecitabine induced differentiation of OCSC and reduced - but did not completely eradicate - OCSC. IL-6 neutralizing antibody (IL-6-Nab) combined with HMA fully eradicated OCSC, and the combination blocked IL-6/IL6-R/pSTAT3-mediated ALDH1A1 expression and eliminated OCSC in residual tumors that persisted in vivo after chemotherapy. We conclude that IL-6 signaling blockade combined with an HMA can eliminate OCSC after platinum treatment, supporting this strategy to prevent tumor recurrence after standard chemotherapy.

// James Haley 1 , Sunil Tomar 1 , Nicholas Pulliam 1 , Sen Xiong 1 , Susan M. Perkins 2 , Adam R. Karpf 3 , Sumegha Mitra 1, 4 , Kenneth P. Nephew 1, 5, 6 , Anirban K. Mitra 1, 5, 7 1 Medical Sciences Program, Indiana University School of Medicine, Bloomington, IN 47405, USA 2 Department of Biostatistics, Indiana University, Indianapolis, IN 46202, USA 3 Eppley Institute and Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE 68198, USA 4 Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202, USA 5 Indiana University Melvin and Bren Simon Cancer Center, Indianapolis, IN 46202, USA 6 Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis, IN 46202, USA 7 Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN 46202, USA Correspondence to: Anirban K. Mitra, email: anmitra@indiana.edu Keywords: ovarian cancer, migration, invasion, proliferation, clonogenicity Received: January 27, 2016 Accepted: April 10, 2016 Published: April 27, 2016 ABSTRACT Genomic analysis of ovarian cancer cell lines has revealed a panel that best represents the most common ovarian cancer subtype, high-grade serous ovarian cancer (HGSOC). However, these HGSOC-like cell lines have not been extensively applied by ovarian cancer researchers to date, and the most commonly used cell lines in the ovarian cancer field do not genetically resemble the major clinical type of the disease. For the HGSOC-like lines to serve as suitable models, they need to be characterized for common functional assays. To achieve that objective, we systematically studied a panel of HGSOC cells CAOV3, COV362, Kuramochi, OVCAR4, OVCAR5, OVCAR8, OVSAHO and SNU119 for migration, invasion, proliferation, clonogenicity, EMT phenotype and cisplatin resistance. They exhibited a range of efficacies and OVCAR5, OVCAR8 and Kuramochi were the most aggressive. SNU119 and OVSAHO cells demonstrated the lowest functional activities. Wide differences in expression of EMT markers were observed between cell lines. SNU119 were the most epithelial and OVCAR8 had the most mesenchymal phenotype. COV362 was the most resistant to cisplatin while CAOV3 was the most sensitive. Taken together, our systematic characterization represents a valuable resource to help guide the application of HGSOC cells by the cancer research community.

Rationale: The stress-induced growth arrest and DNA damage–inducible α (GADD45a) gene is up-regulated by mechanical stress with GADD45a knockout (GADD45a−/−) mice demonstrating both increased susceptibility to ventilator-induced lung injury (VILI) and reduced levels of the cell survival and vascular permeability signaling effector (Akt). However, the functional role of GADD45a in the pathogenesis of VILI is unknown. Objectives: We sought to define the role of GADD45a in the regulation of Akt activation induced by mechanical stress. Methods: VILI-challenged GADD45a−/− mice were administered a constitutively active Akt1 vector and injury was assessed by bronchoalveolar lavage cell counts and protein levels. Human pulmonary artery endothelial cells (EC) were exposed to 18% cyclic stretch (CS) under conditions of GADD45a silencing and used for immunoprecipitation, Western blotting or immunofluoresence. EC were also transfected with mutant ubiquitin vectors to characterize site-specific Akt ubiquitination. DNA methylation was measured using methyl-specific polymerase chain reaction assay. Measurements and Main Results: Studies exploring the linkage of GADD45a with mechanical stress and Akt regulation revealed VILI-challenged GADD45a−/− mice to have significantly reduced lung injury on overexpression of Akt1 transgene. Increased mechanical stress with 18% CS in EC induced Akt phosphorylation via E3 ligase tumor necrosis factor receptor–associated factor 6 (TRAF6)–mediated Akt K63 ubiquitination resulting in Akt trafficking and activation at the membrane. GADD45a is essential to this process because GADD45a-silenced endothelial cells and GADD45a−/− mice exhibited increased Akt K48 ubiquitination leading to proteasomal degradation. These events involve loss of ubiquitin carboxyl terminal hydrolase 1 (UCHL1), a deubiquitinating enzyme that normally removes K48 polyubiquitin chains bound to Akt thus promoting Akt K63 ubiquitination. Loss of GADD45a significantly reduces UCHL1 expression via UCHL1 promoter methylation resulting in increased Akt K48 ubiquitination and reduced Akt levels. Conclusions: These studies highlight a novel role for GADD45a in the regulation of site-specific Akt ubiquitination and activation and implicate a significant functional role for GADD45a in the clinical predisposition to VILI.

Objectives: Osteoporosis is a common condition in postmenopausal women. Bone mineral density (BMD), the major determinant of osteoporotic fracture risk, has a particular genetic background. However, consensus on the association of BMD with specific gene locus has not been reached. The race and ethnicity specific divergence in association studies must be assessed to predict the susceptibility and therapeutic response of associated genes. We investigated the potential association of Vitamin D receptor (VDR) gene polymorphisms ApaI, BsmI, Fok I and TaqI with BMD in 246 postmenopausal Indian women (average age 54.2 ± 3.4 years). Methods: The subjects were genotyped by PCR-RFLP and underwent BMD measurements at spine and hip by dual energy X-ray absorptiometery. Results: The average BMD at spine and hip of women with genotypes aa, bb (presence of restriction sites for ApaI and BsmI), FF and TT (absence of restriction sites for Fok I and TaqI) was more than 10% higher than those with genotypes AA, BB, ff and tt, respectively. The interaction between BsmI, ApaI and TaqI genotypes showed significant effect of BsmI-ApaI genotypes (p = 0.052) in this combination on BMD. However, presence of T allele in combination showed positive influence on BMD. Within the group, genotypes BB, ff and tt were significantly prevalent in women with osteoporotic bone mass, tt and BB had significantly higher adjusted odd ratio (OR) for age more than 55years. Conclusion: Study reveals that VDR gene polymorphisms are associated with BMD in Indian women and perhaps, influence some determinant of bone metabolism. Ethnicity may attribute to frequency variation, however, allele impact remains same. © 2006 Elsevier Ireland Ltd. All rights reserved.

The statins are a class of 3-hydroxy-3-methylglutaryl-coenzyme A-reductase inhibitors that are recognized to have pleiotropic properties. We previously reported the attenuation of LPS-induced murine acute lung injury (ALI) by simvastatin in vivo and identified relevant effects of simvastatin on endothelial cell (EC) signaling, activation, and barrier function in vitro. In particular, simvastatin induces the upregulation of integrin-β4, which in turn inhibits EC inflammatory responses via attenuation of MAPK signaling. The role of integrin-β4 in murine ALI protection by simvastatin, however, is unknown. We initially confirmed a time- and dose-dependent effect of simvastatin on increased integrin-β4 mRNA expression in human lung EC with peak protein expression evident at 16 h. Subsequently, reciprocal immunoprecipitation demonstrated an attenuation of LPS-induced integrin-β4 tyrosine phosphorylation by simvastatin (5 μM, 16 h). Increased expression of EC inflammatory cytokines [IL-6, IL-8, monocyte chemoattractant protein (MCP)-1, regulated on activation normal T cell expressed and secreted (RANTES)] by LPS (500 ng/ml, 4 h) was also significantly attenuated by simvastatin pretreatment (5 μM, 16 h), but this effect was reversed by cotreatment with an integrin-β4-blocking antibody. Finally, although simvastatin (20 mg/kg) conferred significant protection in murine ALI as evidenced by decreased bronchoalveolar lavage fluid cell counts, protein, inflammatory cytokines (IL-6, IL-1β, MCP-1, RANTES), decreased Evans blue dye albumin extravasation in lung tissue, and changes on lung histology, these effects were reversed by the integrin-β4-blocking antibody (IV, 1 mg/kg, 2 h before LPS). These findings support integrin-β4 as an important mediator of ALI protection by simvastatin and implicate signaling by integrin-β4 as a novel therapeutic target in patients with ALI.

We explored the mechanistic involvement of the growth arrest and DNA damage-inducible gene GADD45a in lipopolysaccharide (LPS)- and ventilator-induced inflammatory lung injury (VILI). Multiple biochemical and genomic parameters of inflammatory lung injury indicated that GADD45a−/− mice are modestly susceptible to intratracheal LPS-induced lung injury and profoundly susceptible to high tidal volume VILI, with increases in microvascular permeability and bronchoalveolar lavage levels of inflammatory cytokines. Expression profiling of lung tissues from VILI-challenged GADD45a−/− mice revealed strong dysregulation in the B-cell receptor signaling pathway compared with wild-type mice and suggested the involvement of PI3 kinase/Akt signaling components. Western blot analyses of lung homogenates confirmed ∼50% reduction in Akt protein levels in GADD45a−/− mice accompanied by marked increases in Akt ubiquitination. Electrical resistance measurements across human lung endothelial cell monolayers with either reduced GADD45a or Akt expression (siRNAs) revealed significant potentiation of LPS-induced human lung endothelial barrier dysfunction, which was attenuated by overexpression of a constitutively active Akt1 transgene. These studies validate GADD45a as a novel candidate gene in inflammatory lung injury and a significant participant in vascular barrier regulation via effects on Akt-mediated endothelial signaling.—Meyer, N. J., Huang, Y., Singleton, P. A., Sammani, S., Moitra, J., Evenoski, C. L., Husain, A. N., Mitra, S., Moreno-Vinasco, L., Jacobson, J. R., Lussier, Y. A., Garcia, J. G. N. GADD45a is a novel candidate gene in inflammatory lung injury via influences on Akt signaling.

Bone mineral density (BMD) is the major determinant of osteoporotic fracture risk with a particular genetic background. However, consensus on the association of BMD with specific gene locus has not been reached. In the present study, we investigated the potential association of estrogen receptor alpha (ER alpha) gene intron I polymorphisms with BMD in 246 postmenopausal Indian women (average age 54.2+/-3.4 years). All the subjects were genotyped for XbaI and PvuII polymorphisms and underwent BMD measurements at spine and hip by dual energy X-ray absorptiometery. The average BMD of subjects with the genotypes XX and PP (absence of restriction sites for XbaI and PvuII, respectively) was 12.7 and 5.4% higher at the spine and 13.1 and 4.6% higher at the hip, respectively, than those with genotypes xx and pp. In age vs. BMD scatterplot, the intercept and slope of regression lines for genotypes xx and pp at spine and hip demonstrated comparatively rapid decrease in BMD across the age. The genotype XX was significantly prevalent (p

Rationale: Growth arrest DNA damage inducible alpha (GADD45a) is a stress-induced gene we have shown to participate in the pathophysiology of ventilator-induced lung injury (VILI) via regulation of mechanical stress-induced Akt ubiquitination and phosphorylation. The regulation of GADD45a expression by mechanical stress and its relationship with acute lung injury (ALI) susceptibility and severity, however, remains unknown. Objectives: We examined mechanical stress-dependent regulatory elements (MSRE) in the GADD45a promoter and the contribution of promoter polymorphisms in GADD45a expression and ALI susceptibility. Methods and Results: Initial studies in GADD45a knockout and heterozygous mice confirmed the relationship of GADD45a gene dose to VILI severity. Human lung endothelial cells (EC) transfected with a luciferase vector containing the full length GADD45a promoter sequence (2771 to +223) demonstrated a .4 fold increase in GADD45a expression in response to 18% cyclic stretch (CS, 4 h) compared to static controls while specific promoter regions harboring CS-dependent MSRE were identified using vectors containing serial deletion constructs of the GADD45a promoter. In silico analyses of GADD45a promoter region (2371 to 2133) revealed a potential binding site for specificity protein 1 (SP1), a finding supported by confirmed SP1 binding with the GADD45a promoter and by the significant attenuation of CS-dependent GADD45a promoter activity in response to SP1 silencing. Separately, case-control association studies revealed a significant association of a GADD45a promoter SNP at 2589 (rs581000, G.C) with reduced ALI susceptibility. Subsequently, we found allelic variation of this SNP is associated with both differential GADD45a expression in mechanically stressed EC (18% CS, 4 h) and differential binding site of interferon regulatory factor 7 (IRF7) at this site. Conclusion: These results strongly support a functional role for GADD45a in ALI/VILI and identify a specific gene variant that confers risk for ALI.