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Transforming growth factor-beta (TGF-beta), Smads, and the cyclin-dependent kinase (cdk) inhibitor p21(WAF1) are important in the pathogenesis of diabetic tubular hypertrophy. Phosphoinositide 3 kinase (PI3K)/Akt kinase activity is increased in diabetic glomerular hypertrophy. Thus, we studied the role of PI3K in high glucose (30 mM)-induced p21(WAF1), Smad2/3, and cell cycle-dependent hypertrophy in LLC-PK1 cells. We found that high glucose time-dependently (1-48 h) increased PI3K/Akt kinase activity. LY294002 (a PI3K inhibitor) attenuated high glucose-induced cell cycle-dependent (G(0)/G(1) phase) hypertrophy at 72 h while attenuating high glucose-induced p21(WAF1) gene transcription and protein expression at 36-48 h. LY294002 also attenuated high glucose-induced binding of p21(WAF1) to the cyclin E/cdk2 complex, whereas attenuating high glucose-induced TGF-beta bioactivity, Smad2/3 phosphorylation, and Smad2/3 DNA-binding activity at 36-48 h. We concluded that PI3K is required for high glucose-induced cell cycle-dependent hypertrophy, p21(WAF1) transcription and expression, p21(WAF1) binding to the cyclin E/cdk2 complex, TGF-beta bioactivity, and Smad2/3 activity in LLC-PK1 cells. [ABSTRACT FROM AUTHOR]

The role of long non-coding RNAs in fibroid pathogenesis remains largely unexplored. In a previous study, we found elevated XIST (X-inactive specific transcript) levels in fibroids, which sponged miR-29c and miR-200c, leading to the overexpression of their target genes. This study aimed to assess the therapeutic potential of XIST downregulation in fibroid treatment. Ovariectomized SCID (severe combined immunodeficiency) mice were implanted with fibroid tumors transduced with XIST siRNA or a control via lentivirus. After 1 month, animals were sacrificed and the xenografts were removed for further analysis. XIST knockdown reduced tumor weight by 15% and increased miR-29c and miR-200c expression by 3.9-fold and 2.2-fold, respectively. The mRNA expression of miR-29c targets (COL3A1, TGF-β3, CDK2, SPARC) and miR-200c targets (CDK2, FN1, TDO2), as well as PRL, E2F1, and EZH2, was significantly decreased. Protein abundance of collagen, COL3A1, FN1, CDK2, SPARC, and EZH2 was also reduced. IHC analysis of xenograft sections using the markers of Ki67 for cell proliferation and cleaved caspase 3 for apoptosis showed decreased cell proliferation and no changes in apoptosis in the XIST knockdown xenografts. This analysis also revealed decreased collagen and E2F1 staining nuclei in the XIST knockdown xenografts. These results indicate that downregulation of XIST in fibroids has beneficial therapeutic effects, by reducing tumor growth and the expression of genes involved in cell proliferation, inflammation, and extracellular matrix regulation., (© 2024 The Author(s). The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Objective: To study the differences in immune cell profiles in uterine fibroids (Fibs) and matched myometrium (Myo)., Design: Observational study., Setting: Laboratory study., Patient(s): The study included tissue that was collected from 10 pairs of Fib and matched Myo from women, not on hormonal medications, undergoing hysterectomy and myomectomy., Intervention(s): None., Main Outcome Measure(s): Differences in immune cell and cytokine composition between Fib and matched Myo., Result(s): The mass cytometry analysis indicated that Fibs had a significantly higher number of natural killer (NK) cells, total macrophages, M2 macrophages, and conventional dendritic cells when compared with matched Myo from the same patient. In contrast, Fibs had significantly fewer CD3 and CD4 T cells when compared with Myo. The mass cytometry analysis results did not show any significant difference in the number of resting mast cells. Immunoflurorescent and immunohistochemical imaging confirmed the cytometry by time of flight results, showing a significantly higher number of NK cells, tryptase-positive mast cells indicative of mast cell activation, total macrophages, and M2 cells in Fibs and a significantly lower number of CD3 and CD4 T cells. The cytokine assay revealed significantly increased levels of human interferon α2, interleukin (IL)-1α, and platelet-derived growth factor AA and significantly lower levels of macrophage colony-stimulating factor and IL-1 receptor antagonist in Fib., Conclusion(s): Our results show significant differences in immune cell populations and cytokine levels between Fib and Myo. These differences could account for the increased inflammation in fib and a potential mechanism by which these tumors evade the immune system. These findings provide a foundation for further studies exploring the role of immune cells in Fib development., Competing Interests: Declaration of Interests D.S. has nothing to disclose. T.-D.C. has nothing to disclose. A.A. has nothing to disclose. A.L. has nothing to disclose. O.K. has nothing to disclose., (Copyright © 2024 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Current medical therapies for fibroids have major limitations due to their hypoestrogenic side effects. Based on our previous work showing the activation of NF-kB in fibroids, we hypothesized that inhibiting NF-kB in vivo would result in the shrinkage of tumors and reduced inflammation. Fibroid xenografts were implanted in SCID mice and treated daily with Bay 11-7082 (Bay) or vehicle for two months. Bay treatment led to a 50% reduction in tumor weight. RNAseq revealed decreased expression of genes related to cell proliferation, inflammation, extracellular matrix (ECM) composition, and growth factor expression. Validation through qRT-PCR, Western blotting, ELISA, and immunohistochemistry (IHC) confirmed these findings. Bay treatment reduced mRNA expression of cell cycle regulators ( CCND1 , E2F1 , and CKS2 ), inflammatory markers ( SPARC , TDO2 , MYD88 , TLR3 , TLR6 , IL6 , TNFα , TNFRSF11A , and IL1β ), ECM remodelers ( COL3A1 , FN1 , LOX , and TGFβ3 ), growth factors ( PRL , PDGFA , and VEGFC ), progesterone receptor, and miR-29c and miR-200c. Collagen levels were reduced in Bay-treated xenografts. Western blotting and IHC showed decreased protein abundance in certain ECM components and inflammatory markers, but not cleaved caspase three. Ki67, CCND1, and E2F1 expression decreased with Bay treatment. This preclinical study suggests NF-kB inhibition as an effective fibroid treatment, suppressing genes involved in proliferation, inflammation, and ECM remodeling.

Our previous studies indicated that there is overexpression of MIAT in fibroids and MIAT is a sponge for the miR-29 family in these tumors. The objective of the present study was to determine if the knockdown of MIAT in fibroid xenografts will increase miR-29 levels and reduce the expression of genes targeted by this miRNA such as collagen and cell cycle regulatory proteins in a mouse model for fibroids. Ovariectomized CB-17 SCID/Beige mice bearing estrogen/progesterone pellets were implanted subcutaneously in the flank with equal weight of fibroid explants which had been transduced by lentivirus for either control (empty vector) or MIAT knockdown for four weeks (n=7). Knockdown of MIAT in fibroid xenografts resulted in a 30% reduction of tumor weight and a marked increase in miR-29a, -b, and -c levels in the xenografts. There was reduced cell proliferation and expression of cell cycle regulatory genes CCND1, CDK2, and E2F1 and no significant changes in apoptosis. The xenografts with MIAT knockdown expressed lower mRNA and protein levels of FN1, COL3A1, and TGF-β3, and total collagen protein. Targeting MIAT, which sponges the pro-fibrotic miR-29 family, is an effective therapy for fibroids by reducing cell proliferation and thereby, tumor growth and accumulation of ECM, which is a hallmark of these benign gynecologic tumors., (© 2024 The Author(s).)

Objective: Fibroids are characterized by marked overexpression of tryptophan 2,3 dioxygenase (TDO2). The objective of this study was to determine the effectiveness of in vivo administration of an inhibitor of TDO2 (680C91) on fibroid size and gene expression., Design: Animal and ex vivo human study., Setting: Academic Research Institution., Subjects: Severe combined immunodeficiency mice bearing human fibroid xenografts treated with vehicle and TDO2 inhibitor., Intervention: Daily intraperitoneal administration of 680C91 or vehicle for 2 months and in vitro studies with fibroid explants., Main Outcome Measures: Tumor weight and gene expression profile of xenografts and in vitro mechanistic experiments using fibroid explants., Results: Compound 680C91 was well-tolerated with no effects on blood chemistry and body weight. Treatment of mice with 680C91 resulted in 30% reduction in the weight of fibroid xenografts after 2 months of treatment and as expected lower levels of kynurenine, the byproduct of tryptophan degradation and an endogenous ligand of aryl hydrocarbon receptor (AhR) in the xenografts. The expression of cytochrome P450 family 1 subfamily B member 1 (CYP1B1), transforming growth factor β3 (TGF-β3), fibronectin (FN1), cyclin-dependent kinase 2 (CDK2), E2F transcription factor 1 (E2F1), interleukin 8 (IL-8) and secreted protein acidic and cysteine rich (SPARC) mRNA were lower in the xenografts of mice treated with 680C91 compared with vehicle controls. Similarly, the protein abundance of collagen, FN1, CYP1B1, and SPARC were lower in the xenografts of 680C9- treated mice compared with vehicle controls. Immunohistochemical analysis of xenografts indicated decreased expression of collagen, Ki67 and E2F1 but no significant changes in cleaved caspase 3 expression in mice treated with 680C91. The levels of kynurenine in the xenografts showed a direct correlation with the tumor weight and FN1 levels. In vitro studies with fibroid explants showed a significant induction of CYP1B1, TGF-β3, FN1, CDK2, E2F1, IL8, and SPARC mRNA by tryptophan, which could be blocked by cotreatment with 680C91 and the AhR antagonist CH-223191., Conclusion: The results indicate that correction of aberrant tryptophan catabolism in fibroids could be an effective treatment through its effect to reduce cell proliferation and extracellular matrix accumulation., Competing Interests: Declaration of Interests T.D.C. and O.K. report a patent “Use of Tryptophan 2,3 dioxygenase (TDO2) inhibitors for treatment of fibroids” was applied in 2021. N.T. has nothing to disclose. S.R. has nothing to disclose. D.K. has nothing to disclose. D.B. has nothing to disclose., (Copyright © 2023 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

The objective of this study was to elucidate the expression of long non-coding RNA (lncRNA) in leiomyomas (Lyo) and paired myometrium (Myo) and explore the impact of race and MED12 mutation. Fold change analysis (Lyo/paired Myo) indicated the expression of 63 lncRNAs was significantly altered in the mutated group but not in the non-mutated Lyo. Additionally, 65 lncRNAs exhibited an over 1.5-fold change in the Black but not the White group. Fifteen differentially expressed lncRNAs identified with next-generation sequencing underwent qRT-PCR confirmation. Compared with Myo, the expression of TPTEP1 , PART1 , RPS10P7 , MSC-AS1 , SNHG12 , CA3-AS1 , LINC00337 , LINC00536 , LINC01436 , LINC01449 , LINC02433 , and LINC02624 was significantly higher, while the expression of ZEB2-AS1 , LINC00957 , and LINC01186 was significantly lower. Comparison of normal Myo with diseased Myo showed significant differences in the expression of several lncRNAs. Analysis based on race and Lyo MED12 mutation status indicated a significantly higher expression of RPS10P7 , SNHG12 , LINC01449 , LINC02433 , and LINC02624 in Lyo from Black patients. The expression of TPTEP1 , PART1 , RPS10P7 , MSC-AS1 , LINC00337 , LINC00536 , LINC01436 , LINC01449 , LINC02433 , and LINC02624 was higher, while LINC01186 was significantly lower in the MED12-mutated group. These results indicate that Lyo are characterized by aberrant lncRNA expression, which is further impacted by race and Lyo MED12 mutation status.

The objective of this study was to determine if the aberrant expression of select genes could form the basis for the racial disparity in fibroid characteristics. The next-generation RNA sequencing results were analyzed as fold change [leiomyomas/paired myometrium, also known as differential expression (DF)], comparing specimens from White (n = 7) and Black (n = 12) patients. The analysis indicated that 95 genes were minimally changed in tumors from White (DF ≈ 1) but were significantly altered by more than 1.5-fold (up or down) in Black patients. Twenty-one novel genes were selected for confirmation in 69 paired fibroids by qRT-PCR. Among these 21, coding of transcripts for the differential expression of FRAT2 , SOX4 , TNFRSF19 , ACP7 , GRIP1 , IRS4 , PLEKHG4B , PGR , COL24A1 , KRT17 , MMP17 , SLN , CCDC177 , FUT2 , MYO5B , MYOG , ZNF703 , CDC25A , and CDCA7 was significantly higher, while the expression of DAB2 and CAV2 was significantly lower in tumors from Black or Hispanic patients compared with tumors from White patients. Western blot analysis revealed a greater differential expression of PGR-A and total progesterone (PGR-A and PGR-B) in tumors from Black compared with tumors from White patients. Collectively, we identified a set of genes uniquely expressed in a race/ethnicity-dependent manner, which could form the underlying mechanisms for the racial disparity in fibroids and their associated symptoms.

Tranilast (N-3, 4-dimethoxycinnamoyl anthranilic acid) is an orally administered drug with antiallergic properties and approved in Japan and the Republic of Korea for the treatment of asthma and hypertrophic scars. Previous in vitro studies indicated that tranilast reduced fibroid growth through its inhibitory effects on cell proliferation and induction of apoptosis. The objective of this study was to determine the efficacy of tranilast for treatment of human-derived fibroids in a mouse model. SCID mice (ovariectomized, supplemented with estrogen and progesterone) were implanted with fibroid explants and treated for two months with tranilast (50 m/kg/daily) or the vehicle. After sacrifice, xenografts were excised and analyzed. Tranilast was well tolerated without adverse side effects. There was a 37% reduction in tumor weight along with a significant decrease in staining for Ki67, CCND1, and E2F1; a significant increase in nuclear staining for cleaved caspase 3; and reduced staining for TGF-β3 and Masson's trichrome in the tranilast treated mice. There was a significant inhibition of mRNA and protein expression of fibronectin, COL3A1, CCND1, E2F1, and TGF-β3 in the xenografts from the tranilast-treated mice. These promising therapeutic effects of tranilast warrant additional animal studies and human clinical trials to evaluate its efficacy for treatment of fibroids.

Introduction: Hyperoxia-induced lung injury is characterized by acute alveolar injury, disrupted epithelial-mesenchymal signaling, oxidative stress, and surfactant dysfunction, yet currently, there is no effective treatment. Although a combination of aerosolized pioglitazone (PGZ) and a synthetic lung surfactant (B-YL peptide, a surfactant protein B mimic) prevents hyperoxia-induced neonatal rat lung injury, whether it is also effective in preventing hyperoxia-induced adult lung injury is unknown., Method: Using adult mice lung explants, we characterize the effects of 24 and 72-h (h) exposure to hyperoxia on 1) perturbations in Wingless/Int (Wnt) and Transforming Growth Factor (TGF)-β signaling pathways, which are critical mediators of lung injury, 2) aberrations of lung homeostasis and injury repair pathways, and 3) whether these hyperoxia-induced aberrations can be blocked by concomitant treatment with PGZ and B-YL combination., Results: Our study reveals that hyperoxia exposure to adult mouse lung explants causes activation of Wnt (upregulation of key Wnt signaling intermediates β-catenin and LEF-1) and TGF-β (upregulation of key TGF-β signaling intermediates TGF-β type I receptor (ALK5) and SMAD 3) signaling pathways accompanied by an upregulation of myogenic proteins (calponin and fibronectin) and inflammatory cytokines (IL-6, IL-1β, and TNFα), and alterations in key endothelial (VEGF-A and its receptor FLT-1, and PECAM-1) markers. All of these changes were largely mitigated by the PGZ + B-YL combination., Conclusion: The effectiveness of the PGZ + B-YL combination in blocking hyperoxia-induced adult mice lung injury ex-vivo is promising to be an effective therapeutic approach for adult lung injury in vivo., Competing Interests: Declaration of competing interest Authors Drs. Rehan, Walther, and Waring hold a patent on the composition and methods of administering PPARγ agonists, surfactant peptides, and phospholipids for preventing and treating neonatal lung injury (Patent #:20220047681). No other competing interest or conflict of interest to declare. All authors completed the ICMJE Form., (Copyright © 2023. Published by Elsevier Ltd.)

Recent studies have demonstrated that somatic MED12 mutations in exon 2 occur at a frequency of up to 80% and have a functional role in leiomyoma pathogenesis. The objective of this study was to elucidate the expression profile of coding RNA transcripts in leiomyomas, with and without these mutations, and their paired myometrium. Next-generation RNA sequencing (NGS) was used to systematically profile the differentially expressed RNA transcripts from paired leiomyomas (n = 19). The differential analysis indicated there are 394 genes differentially and aberrantly expressed only in the mutated tumors. These genes were predominantly involved in the regulation of extracellular constituents. Of the differentially expressed genes that overlapped in the two comparison groups, the magnitude of change in gene expression was greater for many genes in tumors bearing MED12 mutations. Although the myometrium did not express MED12 mutations, there were marked differences in the transcriptome landscape of the myometrium from mutated and non-mutated specimens, with genes regulating the response to oxygen-containing compounds being most altered. In conclusion, MED12 mutations have profound effects on the expression of genes pivotal to leiomyoma pathogenesis in the tumor and the myometrium which could alter tumor characteristics and growth potential.

Objective: To determine the expression of enzymes in tryptophan (Trp) catabolism in fibroids and matched myometrium and determine the effects of race and mediator complex subunit 12 gene (MED12) mutation on their expression., Design: Experimental laboratory study., Setting: Academic research laboratory., Patient(s): Women of reproductive age who underwent hysterectomy while on no hormonal medications before surgery., Intervention(s): Fibroids and matched myometrium were obtained from patients who underwent hysterectomy from different race or ethnic groups., Main Outcome Measure(s): The expression of enzymes in the Trp catabolic pathway, tryptophan transporters, and the cytochrome P450 1B1 gene (CYP1B1) in the fibroids and matched myometrium of women from different race and ethnic groups and in tumors bearing the MED12 mutation and tumors without the mutation was determined using quantitative reverse-transcription polymerase chain reaction. The levels of serotonin, kynurenic acid (KYNA), and nicotinamide adenine dinucleotide (NAD) were determined using enzyme-linked immunosorbent assay., Result(s): In fibroids, the expression of tryptophan hydroxylase 1 (TPH1), kynurenine amino transferase (KAT)2, large neutral amino acid transporter small subunit 2 (SLC7A8), and large neutral amino acid transporter small subunit 1 (SLC7A5) messenger RNA (mRNA) was high and that of kynureninase (KYNU) and tryptophanyl-tRNA ligase (WARS1) mRNA was low, with no changes in the expression of WARS2, kynurenine formamidase (AFMID), kynurenine 3-monooxygenase (KMO), KAT1, KAT3, and KAT4 compared with that in the matched myometrium (n = 81). The expression of CYP1B1 mRNA, a marker of the activation of the aryl hydrocarbon receptor, was higher in fibroids. Tumors bearing the MED12 mutation expressed higher levels of CYP1B1 and lower levels of WARS1, KAT1, KAT3, and KAT4 mRNAs compared with tumors without the MED12 mutation. Race or ethnicity affected the expression of KYNU, with tumors from African American and Hispanic patients expressing lower levels of KYNU mRNA compared with those from Caucasian patients. We also quantified the levels of serotonin, KYNA, and NAD, which are the end products of Trp catabolism. There were no significant differences in the levels of serotonin and KYNA, whereas the levels of NAD were lower in fibroids than in the paired myometrium. This reduction in the levels of NAD was independent of race or ethnicity., Conclusion(s): In addition to the expression of tryptophan 2,3-dioxygenase or indoleamine-pyrrole 2,3-dioxygenase, there was marked dysregulation in the expression of other enzymes in the Trp metabolic pathway and Trp transporters in fibroids. Both MED12 mutation status and race or ethnicity had selective effects on the expression of the components of this pathway. Additional functional studies are necessary to establish the physiologic significance of the tryptophan degradation pathway in the pathogenesis of fibroids and its potential as a target for novel therapies., (Copyright © 2022 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Super-enhancer-associated long non-coding RNAs (SE-lncRNAs) are a specific set of lncRNAs transcribed from super-enhancer (SE) genomic regions. Recent studies have revealed that SE-lncRNAs play essential roles in tumorigenesis through the regulation of oncogenes. The objective of this study was to elucidate the expression profile of SE-lncRNAs with concurrent assessment of associated mRNAs in leiomyomas and paired myometrium. Arraystar SE-lncRNAs arrays were used to systematically profile the differentially expressed SE-lncRNAs along with the corresponding SE-regulated protein coding genes in eight leiomyomas and paired myometrium. The analysis indicated 7680 SE-lncRNAs were expressed, of which 721 SE-lncRNAs were overexpressed, while 247 SE-lncRNAs were underexpressed by 1.5-fold or greater in leiomyoma. Thirteen novel SE-lncRNAs and their corresponding protein coding genes were selected, and their expression was confirmed in eighty-one paired leiomyoma tissues by quantitative real-time PCR. The thirteen pairs of SE-lncRNAs and their corresponding protein coding genes included RP11-353N14.2/CBX4, SOCS2-AS1/SOCS2, RP1-170O19.14/HOXA11, CASC15/PRL, EGFLAM-AS1/EGFLAM, RP11-225H22/NEURL1, RP5-1086K13.1/CD58, AC092839.3/SPTBN1, RP11-69I8.3/CTGF, TM4SF1-AS1/TM4SF1, RP11-373D23/FOSL2, RP11-399K21.11/COMTD1, and CTB-113P19.1/SPARC. Among these SE-lncRNAs, the expression of SOCS2-AS1/SOCS2, RP11-353N14.2/CBX4, RP1-170O19.14/HOXA11, and RP11-225H22/NEURL1 was significantly higher in African Americans as compared with Caucasians. The expression of RP11-353N14.2/CBX4, SOCS2-AS1/SOCS2, CASC15/PRL, and CTB-113P19.1/SPARC was significantly higher in tumors with MED12-mutation-positive as compared with MED12-mutation-negative tumors. Collectively, our results indicate that the differential expression of SE in leiomyomas is another mechanism contributing to dysregulation of protein coding genes in leiomyomas and that race and MED12 mutation can influence the expression of a select group of SE., (© 2022. The Author(s).)

The objective of this study was to determine the expression and functional role of a long noncoding RNA (lncRNA) MIAT (myocardial infarction-associated transcript) in leiomyoma pathogenesis. Leiomyoma compared with myometrium (n = 66) expressed significantly more MIAT that was independent of race/ethnicity and menstrual cycle phase but dependent on MED12 (mediator complex subunit 12) mutation status. Leiomyomas bearing the MED12 mutation expressed higher levels of MIAT and lower levels of microRNA 29 family (miR-29a, -b, and -c) compared with MED12 wild-type leiomyomas. Using luciferase reporter activity and RNA immunoprecipitation analysis, MIAT was shown to sponge the miR-29 family. In a 3-dimensional spheroid culture system, transient transfection of MIAT siRNA in leiomyoma smooth muscle cell (LSMC) spheroids resulted in upregulation of miR-29 family and downregulation of miR-29 targets, collagen type I (COL1A1), collagen type III (COL3A1), and TGF-β3 (transforming growth factor β-3). Treatment of LSMC spheroids with TGF-β3 induced COL1A1, COL3A1, and MIAT levels, but repressed miR-29 family expression. Knockdown of MIAT in LSMC spheroids blocked the effects of TGF-β3 on the induction of COL1A1 and COL3A1 expression. Collectively, these results underscore the physiological significance of MIAT in extracellular matrix accumulation in leiomyoma., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objective: To determine the expression and functional roles of indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase (TDO2) in leiomyoma., Design: Experimental study., Setting: Academic research laboratory., Patient(s): Women undergoing hysterectomy for leiomyoma., Intervention(s): Blockade of IDO1 and TDO2., Main Outcome Measure(s): Expression of IDO1 and TDO2 in leiomyoma and the effects of their inhibitors on the extracellular matrix., Result(s): Leiomyoma expressed significantly higher levels of IDO1 and TDO2 messenger ribonucleic acid (mRNA; 60.3%, 35/58 pairs and 98.3%, 57/58 pairs, respectively) and protein (54%, 27/50 pairs and 92%, 46/50 pairs, respectively) as well as the enzyme activity marker kynurenine (78.3%, 36/46 pairs for IDO1/TDO2) compared with levels in matched myometrium. The expression of TDO2 but not IDO1 mRNA was significantly higher in fibroids from African American compared with that in Caucasian and Hispanic patients. The TDO2 but not the IDO1 protein and mRNA levels were more abundant in fibroids bearing the MED12 mutation compared with results in wild-type leiomyomas. Treatment of leiomyoma smooth muscle cell and myometrial smooth muscle cell spheroids with the TDO2 inhibitor 680C91 but not the IDO1 inhibitor epacadostat significantly repressed cell proliferation and the expression of collagen type I (COL1A1) and type III (COL3A1) in a dose-dependent manner; these effects were more pronounced in leiomyoma smooth muscle cells compared with myometrial smooth muscle cell spheroids., Conclusion(s): These results underscore the physiological significance of the tryptophan degradation pathway in the pathogenesis of leiomyomas and the potential utility of anti-TDO2 drugs for treatment of leiomyomas., (Copyright © 2021 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Perinatal smoke/nicotine exposure alters lung development and causes asthma in exposed offspring, transmitted transgenerationally. The mechanism underlying the transgenerational inheritance of perinatal smoke/nicotine-induced asthma remains unknown, but germline epigenetic modulations may play a role. Using a well-established rat model of perinatal nicotine-induced asthma, we determined the DNA methylation pattern of spermatozoa of F1 rats exposed perinatally to nicotine in F0 gestation. To identify differentially methylated regions (DMRs), reduced representation bisulfite sequencing was performed on spermatozoa of F1 litters. The top regulated gene body and promoter DMRs were tested for lung gene expression levels, and key proteins involved in lung development and repair were determined. The overall CpG methylation in F1 sperms across gene bodies, promoters, 5'-UTRs, exons, introns, and 3'-UTRs was not affected by nicotine exposure. However, the methylation levels were different between the different genomic regions. Eighty one CpG sites, 16 gene bodies, and 3 promoter regions were differentially methylated. Gene enrichment analysis of DMRs revealed pathways involved in oxidative stress, nicotine response, alveolar and brain development, and cellular signaling. Among the DMRs, Dio1 and Nmu were the most hypermethylated and hypomethylated genes, respectively. Gene expression analysis showed that the mRNA expression and DNA methylation were incongruous. Key proteins involved in lung development and repair were significantly different (FDR < 0.05) between the nicotine and placebo-treated groups. Our data show that DNA methylation is remodeled in offspring spermatozoa upon perinatal nicotine exposure. These epigenetic alterations may play a role in transgenerational inheritance of perinatal smoke/nicotine induced asthma., (© 2021 Federation of American Societies for Experimental Biology.)

Objective: To determine the expression and functional roles of a long noncoding RNA (lncRNA) X-inactive specific transcript (XIST) in leiomyoma., Design: Experimental study., Setting: Academic research laboratory., Patient(s): Women undergoing hysterectomy for leiomyoma., Intervention(s): Overexpression and underexpression of XIST; blockade of specific protein 1 (SP1)., Main Outcome Measure(s): Expression of XIST in leiomyoma and its effects on microRNA 29c (miR-29c), miR-200c, and their targets., Result(s): Leiomyoma expressed statistically significantly more XIST as compared with matched myometrium, independent of race/ethnicity and menstrual cycle phase. By use of a three-dimensional spheroid culture system, we found reduced XIST levels in leiomyoma smooth muscle cells (LSMC) after treatment with 17β-estradiol, progesterone, and their combination. The expression of XIST was down-regulated by treatment with the SP1-inhibitor mithramycin A and SP1 small interfering RNA. Knockdown of XIST resulted in inhibition of cell proliferation, up-regulation of miR-29c and miR-200c, and a concomitant inhibition of the target genes of these miRNAs, namely collagen type I (COL1A1), collagen type III (COL3A1), and fibronectin (FN1). By contrast, overexpression of XIST in myometrium smooth muscle cells repressed miR-29c and miR-200c, and induced COL1A1, COL3A1, and FN1 levels. By use of RNA immunoprecipitation analysis we confirmed XIST has sponge activity over miR-29c and miR-200c, which is more pronounced in leiomyoma as compared with myometrium., Conclusion(s): Our data demonstrate that increased expression of XIST in leiomyoma results in reduced expression of miR-29c and miR-200c with a consequent up-regulation of the genes targeted by these microRNAs including COL1A1, COL3A1, and FN1, which play key roles in extracellular matrix accumulation associated with fibroids., (Copyright © 2020 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Electronic-cigarette (e-cig) vaping is a serious concern, as many pregnant women who vape consider it safe. However, little is known about the harmful effects of prenatal e-cig exposure on adult offspring, especially on extracellular-matrix (ECM) deposition and myogenesis in the lungs of offspring. We evaluated the biochemical and molecular implications of maternal exposure during pregnancy to e-cig aerosols on the adult offspring of both sexes, with a particular focus on pulmonary ECM remodeling and myogenesis. Pregnant CD-1 mice were exposed to e-cig aerosols with or without nicotine, throughout gestation, and lungs were collected from adult male and female offspring. Compared with the air-exposed control group, female mice exposed to e-cig aerosols, with or without nicotine, demonstrated increased lung protein abundance of LEF-1 (lymphoid enhancer-binding factor 1), fibronectin, and E-cadherin, whereas altered E-cadherin and PPARγ (peroxisome proliferator-activated receptor γ) levels were observed only in males exposed to e-cig aerosols with nicotine. Moreover, lipogenic and myogenic mRNAs were dysregulated in adult offspring in a sex-dependent manner. PAI-1 (plasminogen activator inhibitor-1), one of the ECM regulators, was significantly increased in females exposed prenatally to e-cig aerosols with nicotine and in males exposed to e-cig aerosols compared with control animals exposed to air. MMP9 (matrix metalloproteinase 9), a downstream target of PAI-1, was downregulated in both sexes exposed to e-cig aerosols with nicotine. No differences in lung histology were observed among any of the treatment groups. Overall, adult mice exposed prenatally to e-cig aerosols could be predisposed to developing pulmonary disease later in life. Thus, these findings suggest that vaping during pregnancy is unsafe and increases the propensity for later-life interstitial lung diseases.

Although increased predisposition to cardiac fibrosis and cardiac dysfunction has been demonstrated in the perinatally nicotine-exposed heart, the underlying mechanisms remain unclear. With the use of a well-established rat model and cultured primary neonatal rat cardiac fibroblasts, the effect of perinatal nicotine exposure on offspring heart extracellular matrix deposition and the likely underlying mechanisms were investigated. Perinatal nicotine exposure resulted in increased collagen type I (COL1A1) and III (COL3A1) deposition along with a decrease in miR-29 family and an increase in long noncoding RNA myocardial infarction-associated transcript (MIAT) levels in offspring heart. Nicotine treatment of isolated primary neonatal rat cardiac fibroblasts suggested that these effects were mediated via nicotinic acetylcholine receptors including α7 and the induced collagens accumulation was reversed by a gain-of function of miR-29 family. Knockdown of MIAT resulted in increased miR-29 family and decreased COL1A1 and COL3A1 levels, suggesting nicotine-mediated MIAT induction as the underlying mechanism for nicotine-induced collagen deposition. Luciferase reporter assay and RNA immunoprecipitation studies showed an intense physical interaction between MIAT, miR-29 family, and argonaute 2, corroborating the mechanistic link between perinatal nicotine exposure and increased extracellular matrix deposition. Overall, perinatal nicotine exposure resulted in lower miR-29 family levels in offspring heart, while it elevated cardiac MIAT and collagen type I and III levels. These findings provide mechanistic basis for cardiac dysfunction in perinatal nicotine-exposed offspring and offer multiple novel potential therapeutic targets. NEW & NOTEWORTHY Using an established rat model and cultured primary neonatal cardiac fibroblasts, we show that nicotine mediated MIAT induction as the underlying mechanism for the excessive cardiac collagen deposition. These observations provide mechanistic basis for the increased predisposition to cardiac dysfunction following perinatal cigarette/nicotine exposure and offer novel potential therapeutic targets.

Objective: To determine the mechanism by which tranilast induces miR-200c expression in leiomyoma smooth muscle cells (LSMCs)., Design: Experimental study., Setting: Academic research laboratory., Patient(s): Women undergoing hysterectomy for leiomyoma., Intervention(s): Blockade of RelA/p65., Main Outcome Measure(s): Effects of tranilast and blockade of RelA/p65 on miR-200c expression., Result(s): Tranilast, an inflammation inhibitor, dose-dependently induced miR-200c in LSMCs and myometrium smooth muscle cells (MSMCs), with a more profound effect in LSMCs than in MSMCs. The treatment of LSMCs with Bay 117082, an inhibitor of IκB phosphorylation, further enhanced miR-200c induction by tranilast. The knockdown of RelA/p65 by small interfering RNA also induced miR-200c expression in LSMCs. Although tranilast had no effect on total RelA/p65 protein levels in LSMCs, it significantly induced RelA/p65 phosphorylation at S536 while reducing its activity as well as its nuclear translocation. ChIP assay indicated that tranilast reduces the binding ability of RelA/p65 to miR-200c promoter, resulting in miR-200c induction. Tranilast also inhibited interleukin-8 (IL8) expression in LSMCs. The induction of miR-200c by tranilast partially mediates the inhibitory effect of tranilast on the expression of IL8 and cyclin-dependent kinase 2 in LSMCs., Conclusion(s): Induction of miR-200c by tranilast in LSMCs is mediated through a transcriptional mechanism involving inhibition of the nuclear factor κB signaling pathway. These results highlight the significance of inflammation in the pathogenesis of leiomyoma and the potential utility of antiinflammatory drugs for treatment of leiomyomas., (Copyright © 2019 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To determine the expression of miR-29c and its target gene transforming growth factor-β3 (TGF-β3) in leiomyoma and the mechanisms of their reciprocal regulation., Design: Experimental study., Setting: Academic research laboratory., Patient(s): Women undergoing hysterectomy for leiomyoma., Intervention(s): Overexpression and underexpression of miR-29c; blockade of DNA methyltransferase 1 (DNMT1)., Main Outcome Measure(s): The miR-29c and its target gene TGF-β3 in leiomyoma and the effects of TGF-β3 and blockade of DNMT1 on miR-29c expression., Result(s): Leiomyoma expressed significantly lower levels of miR-29c, but higher expression of TGF-β3 compared with matched myometrium. The expression of TGF-β3 and miR-29c were independent of race/ethnicity. Using 3' untranslated region luciferase reporter assay we confirmed that TGF-β3 is a direct target of miR-29c in leiomyoma smooth muscle cells (LSMCs). Gain-of-function of miR-29c in LSMCs inhibited the expression of TGF-β3 at protein and messenger RNA levels, whereas loss-of-function of miR-29c had the opposite effect. Treatment of LSMCs with TGF-β3 inhibited the expression of miR-29c, whereas it stimulated DNMT1 expression. Knockdown of DNMT1 through transfection with small interfering RNA significantly decreased the expression of TGF-β3, and induced miR-29c expression. Knockdown of DNMT1 also attenuated the inhibitory effect of TGF-β3 on miR-29c expression. Furthermore, we demonstrated that TGF-β3 increased the methylation level of miR-29c promoter in LSMCs., Conclusion(s): There is an inverse relationship in the expression of TGF-β3 and miR-29c in leiomyoma. The TGF-β3 is a direct target of miR-29c and inhibits the expression of miR-29c through an epigenetic mechanism. The cross-talk between miR-29c and TGF-β3 provides a feed forward mechanism of fibrosis in leiomyoma., (Copyright © 2019 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Nicotine inhalation via electronic cigarettes (e-cigs) is an emerging concern. However, little is known about the acute toxicity in the lungs following inhalation of nicotine-containing e-cig aerosols. We hypothesized that acute exposure to aerosolized nicotine causes lung toxicity by eliciting inflammatory and dysregulated repair responses. Adult C57BL/6J mice were exposed 2 h daily for 3 days to e-cig aerosols containing propylene glycol (PG) with or without nicotine. Acute exposure to nicotine-containing e-cig aerosols induced inflammatory cell influx (neutrophils and CD8a + T-lymphocytes), and release of pro-inflammatory cytokines in bronchoalveolar lavage fluid in a sex-dependent manner. Inhalation of e-cig aerosol containing PG alone significantly augmented the lung levels of various homeostasis/repair mediators (PPARγ, ADRP, ACTA2, CTNNB1, LEF1, β-catenin, E-cadherin, and MMP2) in a sex-dependent manner when compared to air controls. These findings were accompanied by an increase in protein abundance and altered gene expression of lipogenic markers (PPARγ, ADRP) and myogenic markers (fibronectin, α-smooth muscle actin and β-catenin), suggesting a dysregulated repair response in mouse lungs. Furthermore, exposure to nicotine containing e-cig aerosols or PG alone differentially affected the release of pro-inflammatory cytokines in healthy and COPD human 3D EpiAirway tissues. Overall, acute exposure to nicotine containing e-cig aerosols was sufficient to elicit a pro-inflammatory response and altered mRNA and protein levels of myogenic, lipogenic, and extracellular matrix markers in mouse lung in a sex-dependent manner. Thus, acute exposure to inhaled nicotine via e-cig leads to dysregulated repair and inflammatory responses, which may lead to airway remodeling in the lungs., Competing Interests: Conflicting financial interests: The authors have declared that no conflict of interest exists.

The objective of this study was to determine whether miR-93, miR-29c, and miR-200c, which we previously reported to be downregulated in leiomyomas, target cell cycle regulatory proteins that influence cell proliferation. Based on TargetScan algorithm 3 cell cycle regulatory proteins namely, E2F transcription factor 1 (E2F1), Cyclin D1 (CCND1) and CDK2 which were predicted to be targets of these miRNAs were  further analyzed. In 30 hysterectomy specimens, we found the expression of E2F1 and CCND1 messenger RNA (mRNA) was increased in leiomyoma as compared to matched myometrium, with no significant changes in CDK2 mRNA levels. There was a significant increase in the abundance of all 3 proteins in leiomyoma in comparison with matched myometrium. Using luciferase reporter assay, we demonstrated E2F1 and CCND1 are targets of miR-93 and CDK2 is a target of miR-29c and miR-200c. We confirmed these findings through transfection studies in which transfection of primary leiomyoma cells with miR-93 resulted in a significant decrease in the expression of E2F1 and CCND1 mRNA and protein levels, whereas knockdown of miR-93 had the opposite effect. Similarly, overexpression of miR-29c and miR-200c in leiomyoma cells inhibited the expression of CDK2 protein and mRNA, whereas knockdown of this microRNAs (miRNA) had the opposite effect. Transfection of miR-29c, miR-200c, and miR-93 in primary leiomyoma cells resulted in a time-dependent inhibition of cell proliferation and cell motility. These results collectively indicate that the 3 miRNAs known to be downregulated in fibroid tumors are critical in regulation of cell proliferation because of their effects on 3 key cell cycle regulatory proteins, which are overexpressed in uterine leiomyomas.

There is no consensus on the most appropriate method for normalizing an individual's electromyography (EMG) signals from walking, cycling and running in the same data collection. The aim of this study was to compare how the magnitude and repeatability of normalization values differ from three normalization methods and to compare their scaling effect in three moderate intensity activities. Three rounds of maximal voluntary isometric contractions (MVICs), sprint cycling and sprint running were performed to obtain normalization values for each method. EMG from five moderate intensity trials of walking, cycling and running were performed and normalized using each normalization value. Normalization values, coefficients of variation, and peak normalized EMG from the three moderate intensity activities were compared across normalization methods. Sprint running resulted in greater normalization values for 6/9 muscles. MVICs produced the lowest variance in 6/9 muscles. Comparing peak normalized signals of interest across normalization methods, there were significant differences in 6/9, 7/9 and 8/9 muscles for walking, cycling and running, respectively. When investigating a combination of walking, cycling and/or running EMG data, sprint running could be used for normalization, due to its simplicity and its ability to produce a larger normalization value, despite lower repeatability., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Considerable epidemiological and experimental evidence supports the concept that the adult chronic lung disease (CLD), is due, at least in part, to aberrations in early lung development in response to an abnormal intrauterine environment; however, the underlying molecular mechanisms remain unknown. We used a well-established rat model of maternal undernutrition (MUN) during pregnancy that results in offspring intrauterine growth restriction (IUGR) and adult CLD to test the hypothesis that in response to MUN, excess maternal glucocorticoids (GCs) program offspring lung development to a CLD phenotype by altering microRNA (miR)-29 expression, which is a key miR in regulating extracellular matrix (ECM) deposition during development and injury-repair. At postnatal day 21 and 5 mo, compared with the control offspring lung, MUN offspring lung miR-29 expression was significantly decreased in conjunction with an elevated expression of multiple downstream target ECM proteins [collagen (COL)1A1, COL3A1, COL4A5, and elastin], at both mRNA and protein levels. Importantly, MUN-induced changes in miR-29 and target gene expressions were at least partially blocked in the lungs of offspring of MUN dams treated with metyrapone, a selective GC synthesis inhibitor. Furthermore, dexamethasone treatment of cultured fetal rat lung fibroblasts significantly induced miR-29 expression along with the suppression of target ECM proteins. These data, along with the previously known role of miR-29 in regulating ECM deposition in vascular tissue in the MUN offspring, suggest miR-29 to be a common mechanistic denominator for the vascular and pulmonary phenotypes in the IUGR offspring, providing a novel potential therapeutic target.

Objective: To determine the expression profile of small noncoding RNAs (sncRNAs) in leiomyoma, which has not been investigated to date., Design: Laboratory-based investigation., Setting: Academic center., Patient(s): Women undergoing hysterectomy for benign indications., Intervention(s): Next-generation sequencing and screening of an sncRNA database with confirmatory analysis by quantitative reverse-transcription polymerase chain reaction (qRT-PCR)., Main Outcome Measure(s): Expression profile of sncRNAs in leiomyoma and matched myometrium., Result(s): Screening our previously determined RNA sequencing data with the sncRNA database resulted in identification of 15 small nuclear (sn) RNAs, 284 small nucleolar (sno) RNAs, 98 Piwi-interacting (pi) RNAs, 152 transfer (t) RNAs, and 45 ribosomal (r) RNAs, of which 15 snoRNAs, 24 piRNAs, 7 tRNAs, and 6 rRNAs were differentially expressed at a 1.5-fold change cutoff in leiomyoma compared with myometrium. We selected 5 snoRNAs, 4 piRNAs, 1 tRNA, and 1 rRNA that were differentially expressed and confirmed their expression in paired tissues (n = 20) from both phases of the menstrual cycle with the use of qRT-PCR. The results indicated up-regulation of the snoRNAs (SNORD30, SNORD27, SNORA16A, SNORD46, and SNORD56) and down-regulation of the piRNAs (piR-1311, piR-16677, piR-20365, piR-4153), tRNA (TRG-GCC5-1), and rRNA (RNA5SP202) expression in leiomyoma compared with myometrium (P<.05). The pattern of expression of these sncRNAs was similar to RNA sequencing analysis, with no menstrual cycle-dependent differences detected except for SNORD30. Because Argonaute 2 (AGO2) is required for sncRNA-mediated gene silencing, we determined its expression and found greater abundance in leiomyoma., Conclusion(s): Our results provide the first evidence for the differential expression of additional classes of sncRNAs and AGO2 in leiomyoma, implicating their roles as a gene regulatory mechanism., (Copyright © 2018 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

The objective of this study was to identify the expression profile of long noncoding RNAs (lncRNAs) with concurrent assessment of microRNA (miRNA) and messenger RNA (mRNA) profiles in leiomyomas and paired myometrium using next-generation RNA sequencing and assembly of RNA transcripts. Total RNA was isolated from leiomyoma and paired myometrium (N = 8) and samples from 3 pairs were subjected to RNA sequencing. Normalized assembly of over 48 000 lncRNAs resulted in identification of 45 936 lncRNAs. Of these lncRNAs, 22 148 representing overlapping, intergenic, intronic, and antisense subtypes were expressed in all paired tissues, with 5941 (2813 up- and 3128 downregulated at ≥1.5 fold) differentially expressed in leiomyomas. Concurrent RNA sequencing revealed the expression of 2588 miRNAs and 21 814 mRNAs, of which 392 miRNAs and 16 559 mRNAs were expressed in all paired tissues. Of these transcripts, 56 and 92 miRNAs and 2030 and 1825 mRNAs were up- or downregulated at ≥1.5 fold, respectively, in leiomyoma as compared to myometrium. Using quantitative reverse transcription-polymerase chain reaction (QRT-PCR), we confirmed the expression of hepatocellular carcinoma upregulated (HULC), lnc-maternally expressed 3 (MEG3), long intergenic ncRNA 890 (LINC00890), TSIX, long intergenic ncRNA 473 (LINC00473), lnc-KLF9-1, and lnc-POTEM-3 (lncRNA-ATB) in leiomyoma and matched myometrium (N = 8). Collectively, the results presented here provide a comprehensive expression profile of lncRNAs in leiomyomas with concurrent integrated expression of miRNAs and mRNAs and implicate potential regulatory functions of lncRNAs through interactions with specific miRNAs and mRNAs which are known to be critical in the pathogenesis of leiomyoma.

Aims: The objective of this study was to determine the underlying mechanism by which glucocorticoids (GCs) induce of miR-29c expression in vascular smooth muscle cells., Main Methods: QRT-PCR was used for miR-29c detection. Protein levels were determined by western blotting. Knockdown of SP1, DNMT1 and DNMT3A was achieved through transfection with their specific respective siRNAs. The effect of GCs on SP1 activity was determined by luciferase reporter assay and the methylation status in miR-29c promoter was detected by methylation specific PCR. CHIP assay was used to determine the binding ability of SP1 and glucocorticoid receptor (GR) in miR-29c promoter., Key Findings: Treatment of RASMC with SP1 siRNA or SP1 inhibitor, mithramycin A, as well as DNMT1 and DNMT3A siRNAs and an inhibitor of DNMTs, Decitabine, resulted in increased expression of miR-29c (P<0.05). Treatment RASMC with dexamethasone (DEX, 0.1μM) for 24h reduced the expression of SP1 and phosphorylated SP1 at threonine 453 protein levels, repressed SP1 activity, and inhibited the expression of DNMT1 and DNMT3A proteins (P<0.05). Treatment with mifepristone, a GR antagonist, blocked the inhibitory effect of DEX on DNMT1 and DNMT3A protein expression. DEX also suppressed SP1 binding ability in miR-29c promoter and inhibited methylation of miR-29c promoter (P<0.05). Treatment of RASMC with DEX (0.1μM) significantly (P<0.05) increased the binding of GR to the miR-29c promoter., Significance: The stimulatory effect of GCs on miR-29c expression is mediated by these three mechanisms: transcriptionally regulated by SP1, and epigenetically through a methylation-dependent process and GR., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Tranilast (N-3,4-dimethoxycinnamoyl anthranilic acid) is an antiallergic agent with inhibitory effects on cell proliferation and extracellular matrix production. Here we assess the effect of tranilast on the expression of miR-29c and genes functionally involved in cell proliferation, fibrosis, and epigenetic regulation in isolated leiomyoma smooth muscle cells (LSMC). Tranilast significantly inhibited the rate of LSMC proliferation, which was associated with downregulation of cell cycle progression genes cyclin D1 (CCND1) and cyclin-dependent kinase 2 (CDK2) expression at messenger RNA and protein levels ( P < .05). Tranilast also suppressed the expression of collagen type I (COL1), collagen type III alpha 1 chain (COL3A1), the profibrotic cytokine, transforming growth factor β-3 (TGF-β3), DNA (cytosine-5)-methyltransferase 1 (DNMT1), and enhancer of zeste homolog 2 (EZH2), which regulate epigenetic status of gene promoters ( P < .05). Tranilast also significantly induced the expression of cellular and secreted miR-29c through downregulation of methylation status of miR-29c promoter ( P < .05). In addition, tranilast suppressed the activity of luciferase reporter containing 3'UTR of COL3A1 and CDK2, which are downstream targets of miR-29c ( P < .05). Knockdown of miR-29c expression attenuated the inhibitory effects of tranilast on COL3A1 and CDK2 protein expression ( P < .05). Collectively, these findings suggest that tranilast could have therapeutic potential as an inhibitory agent for leiomyoma growth and its associated symptoms.

Objective: To determine the expression of miR-29c and its target genes in leiomyoma and the role of NF-κB, specific protein 1 (SP1), and DNA methylation in its regulation., Design: Experimental study., Setting: Academic research laboratory., Patient(s): Women undergoing hysterectomy for leiomyoma., Intervention(s): Over- and underexpression of miR-29c; blockade of transcription factors., Main Outcome Measure(s): MiR-29c and its target gene levels in leiomyoma and the effects of blockade of transcription factors on miR-29c expression., Result(s): Leiomyoma as compared with myometrium expressed significantly lower levels of miR-29c, with an inverse relationship with expression of its targets, COL3A1 and DNMT3A. Gain of function of miR-29c inhibited the expression of COL3A1 and DNMT3A at protein and mRNA levels, secreted COL3A1, and rate of cell proliferation. Loss of function of miR-29c had the opposite effect. E2, P, and their combination inhibited miR-29c in leiomyoma smooth muscle cells (LSMC). Phosphorylated NF-κB (p65) and SP1 protein expression were significantly increased in leiomyoma. SiRNA knockdown of SP1 and DNMT3A or their specific inhibitors significantly increased the expression of miR-29c, accompanied by the inhibition of cellular and secreted COL3A1 in siRNA-treated cells. Knockdown of p65 also induced miR-29c expression but had no effect on COL3A1 expression., Conclusion(s): MiR-29c expression is suppressed in leiomyoma, resulting in an increase in expression of its targets COL3A1 and DNMT3A. The suppression of miR-29c in LSMC is primarily mediated by SP1, NF-κB signaling, and epigenetic modification. Collectively, these results indicate a significant role for miR-29c in leiomyoma pathogenesis., (Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

The purpose of this study was to examine the hypothesis that excess maternal glucocorticoids in response to maternal undernutrition programs the expression of extracellular matrix (ECM) components potentially by miR-29c. We measured the expression of mRNA (qRT-PCR) and protein (Western blot) for collagen 3A1, collagen 4A5 and matrix metalloproteinase 2 (MMP2) in offspring carotid arteries from three groups of dams: 50% food-restricted in latter half of gestation [maternal undernutrition (MUN)], MUN dams who received metyrapone (MET) (500 mg/ml ) in drinking water from day 10 of gestation to term, and control dams fed an ad libitum diet. The expression of miR-29c was significantly decreased at 3 weeks, 3 months and 9 months in MUN carotid arteries, and these decreases in expression were partially blocked by treatment of dams with MET. The expression pattern of ECM genes that are targets of miR-29c correlated with miR-29c expression. Expression of mRNA was increased for elastin (ELN) and MMP2 mRNA in 3-week MUN carotids; in 9-month carotids there were also significant increases in expression of Col3A1 and Col4A5. These changes in mRNA expression of ECM genes at 3 weeks and 9 months were blocked by MET treatment. Similarly, the expression of ELN and MMP2 proteins at 3 weeks were increased in MUN carotids, and by 9 months there were also increases in expression of Col3A1 and Col4A5, which were blocked by MET in MUN carotids. Overall, the results demonstrate a close correlation between expression of miR-29c and the ECM proteins that are its targets thus supporting our central hypothesis., Competing Interests: Conflict of Interests None.

Maternal undernutrition increases maternal glucocorticoids (GCs) and alters microRNA expression in offspring. Given that the mechanisms of GC action on vascular development are not clear, this study examined the influence of GCs on microRNA 29c (miR-29c) and its predicted targets in primary rat aorta smooth muscle cells (RAOSMCs). Dexamethasone (Dex) and corticosterone (Cor) time-dependently increased miR-29c expression and reduced collagen type III (Col3A1), collagen type IV (Col4A5), elastin (ELN), and matrix metalloproteinase-2 (MMP2) protein in RAOSMCs. These effects were blocked by mifepristone. These genes were also targeted by miR-29c, as confirmed by a significant decrease in luciferase reporter activity of Col3A1 (34%), Col4A5 (45%), ELN (17%), and MMP2 (28%). In cells transfected with reporter plasmids, including the 3'-untranslated region of genes targeted by miR-29c, treatment with Dex or Cor also resulted in decreases in luciferase activity. Gain or loss of function of miR-29c significantly altered mRNA expression of Col3A1 (26% and 26%, respectively), Col4A5 (28% and 32%, respectively), and MMP2 (24% and 14%, respectively) but did not affect ELN. Gain or loss of function of miR-29c also significantly altered protein levels of Col3A1 (51% and 16%, respectively), Col4A5 (56% and 22%, respectively), ELN (53% and 71%, respectively), and MMP2 (28% and 53%, respectively). Coincubation of anti-miR-29c with Dex or Cor partially attenuated the effects of these steroids on protein expression of Col3A1 (25% and 24%, respectively), Col4A5 (26% and 44%, respectively), ELN (31% and 55%, respectively), and MMP2 (46% and 26%, respectively) in RAOSMCs compared with anti-miR negative controls. Our results demonstrate that GCs regulate the expression of Col3A1, Col4A5, ELN, and MMP2, at least in part, through induction of miR-29c., (Copyright © 2015 the American Physiological Society.)

Uterine leiomyosarcoma is a relatively rare malignancy with high mortality due to metastasis and chemoresistance. Leiomyosarcomas share similar morphological characteristics with leiomyomas which are considered to have the potential of transformation into leiomyosarcoma. Accumulated evidence suggests that microRNAs acting as regulators of gene expression at the posttranscriptional level play key roles in diverse biological processes including cellular transformation and tumorigenesis. We hypothesized that miR-200c, whose expression is altered in leiomyomas, equally plays a key role in pathogenesis of leiomyosarcoma. Using SK-LMS-1 leiomyosarcoma cell line as an in vitro model here, we found that the level of expression of miR-200c was significantly lower as compared to isolated leiomyoma smooth muscle cells. Overexpression (gain-of-function) of miR-200c in SK-LMS-1 through direct interaction with 3'-untranslated region of IKBKB, IL8, CDK2, and CCNE2, respectively, resulted in suppression of their expression as determined by quantitative polymerase chain reaction and Western blot analysis. Additionally, gain-of-function of miR-200c through inhibition of IKBKB expression resulted in decreased p65 transcriptional activity in IL8 promoter. Gain-of-function of miR-200c also increased SK-LMS-1 caspase 3/7 activity and inhibited their proliferation and migration. In summary, the results suggest that a progressive decline in miR-200c expression which alters transcriptional regulation of specific target genes that control nuclear factor-κB signaling pathway, inflammation, cell cycle, and migration, in part may promote development and progression of leiomyosarcomas, including their transformation from leiomyomas., (© The Author(s) 2014.)

Objective: To determine the effects of maternal undernutrition (MUN) on the reproductive axis of aging offspring., Design: Animal (rat) study., Setting: Research laboratory., Animal(s): Female Sprague-Dawley rats., Intervention(s): Food restriction during the second half of pregnancy in rats., Main Outcome Measure(s): Circulating gonadotropins, antimüllerian hormone (AMH), ovarian morphology, estrous cyclicity, and gene expression studies in the hypothalamus and ovary in 1-day-old (P1) and aging adult offspring., Result(s): Offspring of MUN dams had low birth weight (LBW) and by adult age developed obesity. In addition, 80% of adult LBW offspring had disruption of estrous cycle by 8 months of age, with the majority of animals in persistent estrous. Ovarian morphology was consistent with acyclicity, with ovaries exhibiting large cystic structures and reduced corpora lutea. There was an elevation in circulating T, increased ovarian expression of enzymes involved in androgen synthesis, an increase in plasma LH/FSH levels, a reduction in E2 levels, and no changes in AMH in adult LBW offspring compared with in control offspring. Hypothalamic expression of leptin receptor (ObRb), estrogen receptor-α (ER-α), and GnRH protein was altered in an age-dependent manner with increased ObRb and ER-α expression in P1 LBW hypothalami and a reversal of this expression pattern in adult LBW hypothalami., Conclusion(s): Our data indicate that the maternal nutritional environment programs the reproductive potential of the offspring through alteration of the hypothalamic-pituitary-gonadal axis. The premature reproductive senescence in LBW offspring could be secondary to the development of obesity and hyperleptinemia in these animals in adult life., (Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

To test the hypothesis that inhibition of offspring angiogenesis by maternal undernutrition (MUN) is mediated by maternal glucocorticoids, 3 groups of dams were studied: controls received ad libitum food; MUN dams were food restricted by 50% from day 10 of gestation; and metyrapone (MET) dams were food restricted and treated with 0.5 mg/mL of MET, a glucocorticoid synthesis inhibitor. The MUN reduced birth weights, reduced vascular endothelial growth factor (VEGF) abundance in P1 aortas, reduced VEGF and VEGF-R2 abundances in P1 mesenteric arterioles, reduced arteriolar endothelial nitric oxide synthase abundance, reduced microvessel density in the anterior tibialis, reduced endothelial cell branching in culture, reduced arteriolar immunoreactivity for proliferating cell nuclear antigen (PCNA), increased active caspase 3 in P1 mesenteric arterioles, and decreased matrix metalloproteinase (MMP)-2 and MMP-9 abundances in lysates of P1 aortas. All of these effects were prevented by treatment with metyrapone. Collectively, these findings suggest that reduced angiogenesis in MUN offspring involves direct inhibitory effects of maternal glucorticoid on fetal VEGF and its receptors.

We have previously reported that leiomyoma expressed lower levels of miR-200c and elevated IL8 as compared to paired myometrium. Here we addressed the regulatory functions of miR-200c on the expression of inflammatory mediators and cellular viability using leiomyomas and paired myometrium and their isolated primary smooth muscle cells. Our results indicated that gain-of function or knockdown of miR-200c in leiomyoma smooth muscle cells (LSMC) regulated IL8 mRNA and protein expression through direct targeting of IKBKB and alteration of NF-kB activity. Additionally, leiomyoma expressed higher levels of phosphorylated IKBKB with no significant difference in the level of IKBKB mRNA and protein as compared to matched myometrium. Gain-of function of miR-200c in LSMC resulted in decreased IkBα phosphorylation and p65 nuclear translocation, which led to decreased p65 transcriptional activity of IL8 promoter, and increased caspase 3/7 activity which was not reversible following IL8 restoration. Collectively, our results suggest that NF-κB signaling pathway is a target of miR-200c regulatory function, and low level of miR-200c expression in leiomyoma by transcriptional regulation of inflammatory mediators such as IL8, in part account for development of leiomyomas.

Purpose: The role of microRNA (miRNA) regulation in corneal wound healing and scar formation has yet to be elucidated. This study analyzed the miRNA expression pattern involved in corneal wound healing and focused on the effect of miR-133b on expression of several profibrotic genes., Methods: Laser-ablated mouse corneas were collected at 0 and 30 minutes and 2 days. Ribonucleic acid was collected from corneas and analyzed using cell differentiation and development miRNA PCR arrays. Luciferase assay was used to determine whether miR-133b targeted the 3' untranslated region (UTR) of transforming growth factor β1 (TGFβ1) and connective tissue growth factor (CTGF) in rabbit corneal fibroblasts (RbCF). Quantitative real-time PCR (qRT-PCR) and Western blots were used to determine the effect of miR-133b on CTGF, smooth muscle actin (SMA), and collagen (COL1A1) in RbCF. Migration assay was used to determine the effect of miR-133b on RbCF migration., Results: At day 2, 37 of 86 miRNAs had substantial expression fold changes. miR-133b had the greatest fold decrease at -14.33. Pre-miR-133b targeted the 3' UTR of CTGF and caused a significant decrease of 38% (P < 0.01). Transforming growth factor β1-treated RbCF had a significant decrease of miR-133b of 49% (P < 0.01), whereas CTGF, SMA, and COL1A1 had significant increases of 20%, 54%, and 37% (P < 0.01), respectively. The RbCF treated with TGFβ1 and pre-miR133b showed significant decreases in expression of CTGF, SMA, and COL1A1 of 30%, 37%, and 28% (P < 0.01), respectively. Finally, there was significant decrease in migration of miR-133b-treated RbCF., Conclusions: Significant changes occur in key miRNAs during early corneal wound healing, suggesting novel miRNA targets to reduce scar formation.

A number of microRNAs (miRNAs), including miR-200 family, are aberrantly expressed in endometriosis and endometrial cancer. Here we assessed the expression and functional aspects of miR-200c in endometrial tissues (N = 52) from normal endometrial biopsies (N = 15), endometrial tissues including those exposed to hormonal therapies (N = 20), and grade I-III endometrial cancer (N = 17). miR-200c expression was elevated in normal endometrial biopsies from mid- and late-luteal phase, and in endometrial tumors as compared to endometrial tissues from peri- and postmenopausal period (P < .05) and its pattern of temporal expression displayed an inverse relationship with the expression of ZEBs. The expression of E-cadherin (CDH1) varied, but expressed at low levels, specifically in endometrial tissues and endometrial tumors. The endometrial expression of ZEBs and CDH1 in patients who were exposed to Depo-Provera and gonadotropin-releasing hormone agonist GnRHa displayed a trend toward lower expression as compared to proliferative phase; however, treatment of Ishikawa cells with 17β-estradiol, progesterone, and medroxy progesterone acetate had modest effects on the expression of miR-200c and ZEBs without affecting CDH1 expression. Gain of function of miR-200c in Ishikawa cells repressed ZEBs, as well as VEGFA, FLT1, IKKβ, and KLF9 expression at transcriptional and translational levels through direct interaction with their respective 3'untranslated regions and increased the rate of their proliferation. These results indicated that endometrial miR-200c expression undergoes dynamic changes during transition from normal into cancerous states; possibly influenced by hormonal milieu and by targeting the expression of specific genes with key regulatory functions in cellular transformation, inflammation, and angiogenesis may influence these events during normal and disease progression.

MicroRNA-200c (miR-200c) through repression of specific target genes has been associated with cellular transition, tumorigenesis, and tissue fibrosis. We explored the expression and functional aspects of miR-200c in genesis of leiomyomas (LYO), benign uterine tumors with fibrotic characteristic. Using LYO and matched myometrium (MYO; n=76) from untreated and from patients exposed to hormonal therapies (GNRH agonist (GNRHa), Depo-Provera, and oral contraceptives), we found that miR-200c was expressed at significantly lower levels (P<0.05) in LYO as compared with MYO. These levels were lower in LYO from African Americans as compared with Caucasians, patients experiencing abnormal uterine bleeding and those exposed to GNRHa therapy. Gain-of-function of miR-200c in isolated leiomyoma smooth muscle cells (LSMCs), myometrial smooth muscle cells (MSMCs), and leiomyosarcoma cell line (SKLM-S1) repressed ZEB1/ZEB2 mRNAs and proteins, with concurrent increase in E-cadherin (CDH1) and reduction in vimentin expression, phenotypic alteration, and inhibition of MSMC and LSMC proliferations. We further validated TIMP2, FBLN5, and VEGFA as direct targets of miR-200c through interaction with their respective 3' UTRs, and other genes as determined by microarray analysis. At tissue levels, LYO expressed lower levels of TIMP2 and FBLN5 mRNAs but increased protein expressions, which to some extent altered due to hormonal exposure. Given the regulatory functions of ZEBs, VEGFA, FBLN5, and TIMP2 on cellular activities that promote cellular transition, angiogenesis, and matrix remodeling, we concluded that altered expression of miR-200c may have a significant impact on the outcome of LYO growth, maintenance of their mesenchymal and fibrotic characteristics, and possibly their associated symptoms.

Context: Evidence suggests that a number of microRNA (miRNA) are aberrantly expressed in endometrial disorders with potential posttranscriptional regulation of their specific target genes, including ovarian steroid receptors., Objectives: Our objective was to assess the endometrial expression of miR-98 and miR-181a and their respective target genes, progesterone (P4) receptor membrane component 1 (PGRMC1) and P4 receptor (PGR)., Design, Setting, and Patients: We evaluated tissue expression and in vitro regulation at an academic university medical center in endometrial biopsies and endometrial tissues from follicular and luteal phases with and without exposure to hormonal therapies and grade I-III endometrial cancer (n = 52)., Interventions: INTERVENTIONS included endometrial biopsies and in vitro transfection., Main Outcome Measures: We evaluated expression and function of miR-98 and miR-181a., Results: Aberrant expression of miR-98 and miR-181a is associated with endometrial transition from normal into cancerous states, which to some extent is influenced by hormonal milieu, and exhibited an inverse relationship with PGMRC1 and PGR expression, respectively. Treatments of Ishikawa cells with 17β-estradiol, P4, or medroxyprogesterone acetate had limited effects on miR-98, miR-181a, and PGRMC1 expression, whereas 17β-estradiol treatment increased PGR expression. In Ishikawa cells, gain of function of miR-98 repressed PGRMC1 and CYP19A1, and miR-181a repressed PGR, DDX3X, and TIMP3 at mRNA and protein levels through direct interactions with their respective 3'-untranslated regions and CCNE1 through miR-181a-induced DDX3X repression, with miR-98 reducing the rate of cell proliferation as compared with controls., Conclusion: miR-98 and miR-181a through their regulatory functions on PGRMC1, PGR, CYP19A1, TIMP3, and DDX3X expression may influence a wide range of endometrial cellular activities during normal menstrual cycle and transition into disease states, including endometrial cancer.

miR-93/106b and their host gene minichromosome maintenance complex component 7 (MCM7) reside at chr7q22, a region frequently rearranged in leiomyomas. We explored the expression of miR-93/106b in leiomyoma and paired myometrium (n = 63) from untreated and patients exposed to hormonal therapies (GnRH agonist, Depo-Provera, and oral contraceptives) from African-Americans and Caucasians and their regulatory functions in isolated paired (n = 15) leiomyoma and myometrial smooth muscle cells and the leiomyosarcoma cell line. At tissue level leiomyomas expressed significantly lower levels of miR-93 and elevated MCM7 as compared with myometrium with limited racial influence or hormonal exposure on their expression. Assessing the regulatory function of miR-93/106b through doxycycline-inducible lentiviral transduction in a microarray analysis, tissue factor (F3) and IL8 were identified as their possible targets. At the tissue level, leiomyomas expressed a significantly lower level of F3 and an elevated IL-8 level, which exhibited an inverse relationship with miR-93 but with limited racial or hormonal influences. The gain of function of miR-93/106b in leiomyoma smooth muscle cells, myometrial smooth muscle cells, and the leiomyosarcoma cell line dose dependently repressed F3 and IL8 through direct interactions with their respective 3'-untranslated region and indirectly through F3 repression inhibited IL8, CTGF, and PAI-1 expression, confirmed by using small interfering RNA silencing or factor Vlla (FVIIa) activation of F3, as well as reducing the rate of proliferation, while increasing caspase-3/7 activity. We concluded that differential expression of miR-93/106b and their direct and/or indirect regulatory functions on F3, IL8, CTGF, and PAI-1 expression, with key roles in inflammation and tissue turnover may be of significance in the outcome of leiomyoma growth and associated symptoms.

Cellular prostatic acid phosphatase (cPAcP), an authentic tyrosine phosphatase, is proposed to function as a negative growth regulator of prostate cancer (PCa) cells in part through its dephosphorylation of ErbB-2. Nevertheless, the direct interaction between cPAcP and ErbB-2 has not been shown nor the specific dephosphorylation site of ErbB-2 by cPAcP. In this report, our data show that the phosphorylation level of ErbB-2 primarily at Tyr(1221/2) correlates with the growth rate of both LNCaP and MDA PCa2b human PCa cells. Further, cPAcP reciprocally co-immunoprecipitated with ErbB-2 in a non-permissive growth condition. Expression of wild type cPAcP, but not inactive mutant, by cDNA in cPAcP-null LNCaP C-81 cells results in decreased tyrosine phosphorylation of ErbB-2 including Tyr(1221/2). Concurrently, Tyr(317) phosphorylation of p52(Shc), proliferating cell nuclear antigen expression, and cell growth are decreased in these cells. Conversely, decreased cPAcP expression by short hairpin RNA in LNCaP C-33 cells was associated with elevated phosphorylation of ErbB-2 initially at Tyr(1221/2). Its downstream p52(Shc), ERK1/2, Akt, Src, STAT-3, and STAT-5 were activated, and cell proliferation, proliferating cell nuclear antigen, and cyclin D1 expression were increased. Stable subclones of C-33 cells by small interfering PAcP had elevated Tyr(1221/2) phosphorylation of ErbB-2 and exhibited androgen-independent growth and increased tumorigenicity in xenograft female animals. In summary, our data together indicate that in prostate epithelia, cPAcP interacts with and dephosphorylates ErbB-2 primarily at Tyr(1221/2) and hence blocks downstream signaling, leading to reduced cell growth. In PCa cells, decreased cPAcP expression is associated with androgen-independent cell proliferation and tumorigenicity as seen in advanced hormone-refractory prostate carcinomas.

The main objectives of the present study were to investigate the chemical reduction of nitrate or nitrite species by zero-valent iron nanoparticle (ZVIN) in aqueous solution and related reaction kinetics or mechanisms using fine structure characterization. This work also exemplifies the utilization of field emission-scanning electron microscope (FE-SEM), transmission electron microscopy (TEM), and x-ray diffraction (XRD) to reveal the speciation and possible reaction pathway in a very complex adsorption and redox reaction process. Experimentally, ZVIN of this study was prepared by sodium borohydride reduction method at room temperature and ambient pressure. The morphology of as-synthesized ZVIN shows that the nearly ball and ultrafine particles ranged of 20-50 nm were observed with FE-SEM or TEM analysis. The kinetic model of nitrites or nitrates reductive reaction by ZVIN is proposed as a pseudo first-order kinetic equation. The nitrite and nitrate removal efficiencies using ZVIN were found 65-83% and 51-68%, respectively, based on three different initial concentrations. Based on the XRD pattern analyses, it is found that the quantitative relationship between nitrite and Fe(III) or Fe(II) is similar to the one between nitrate and Fe(III) in the ZVIN study. The possible reason is due to the faster nitrite reduction by ZVIN. In fact, the occurrence of the relative faster nitrite reductive reaction suggested that the passivation of the ZVIN have a significant contribution to iron corrosion. The extended x-ray absorption fine structure (EXAFS) or x-ray absorption near edge structure (XANES) spectra show that the nitrites or nitrates reduce to N 2 or NH 3 while oxidizing the ZVIN to Fe 2 O 3 or Fe 3 O 4 electrochemically. It is also very clear that decontamination of nitrate or nitrite species in groundwater via the in-situ remediation with a ZVIN permeable reactive barrier would be environmentally attractive.

The cyclin-dependent kinase inhibitor p21(WAF1) is required for diabetic glomerular hypertrophy. High glucose-induced hypertrophy in proximal tubule cells is dependent on transforming growth factor-beta (TGF-beta). Many of the TGF-beta-induced effects are dependent on Smad2/3. Thus, the molecular mechanisms of high glucose-induced p21(WAF1) and hypertrophy were studied in high glucose-cultured proximal tubule-like LLC-PK(1) cells. We found that high glucose (30 mM) induced hypertrophy at 72 h. High glucose also increased the expression of p21(WAF1) protein and p21(WAF1) mRNA transcription and abundance at 48 h. The DNA element in the 5' regulatory region of p21(WAF1) gene essential for high glucose-induced p21(WAF1) gene transcription was identified as Sp1 by a series of the 5' regulatory region of p21(WAF1) gene deletion mutants. Moreover, high glucose activated Smad2/3 while increasing the Sp1 DNA-binding activity. High glucose also increased the Sp1-dependent transcriptional activity of p21(WAF1) gene. High glucose-induced hypertrophy was attenuated by p21(WAF1) short interfering RNA and Smad3 dominant-negative plasmid transfection. We concluded that high glucose induced hypertrophy via Sp1-Smad2/3-dependent activation of p21(WAF1) gene transcription in LLC-PK(1) cells., ((c) 2007 Wiley-Liss, Inc.)

High glucose inhibits endothelial cell proliferation. Thus, we studied cyclin-dependent kinase inhibitor p15(INK4b) in high glucose-induced effects in human umbilical endothelial cells at 24h. High glucose decreased cell proliferation while arresting cells in G(0)/G(1) phase of the cell cycle. High glucose increased phospho-extracellular signal regulated kinase (ERK)1/2, p15(INK4b) protein and mRNA expression. High glucose-inhibited cell proliferation was attenuated by antisense p15(INK4b) oligonucleotide. Moreover, PD98059 attenuated high glucose-induced p15(INK4b) protein expression. High glucose increased transforming growth factor-beta (TGF-beta) gene transcriptional activity and mRNA expression. However, neither SB431542 (type I TGF-beta receptor blocker) nor TGF-beta1 antibody affected high glucose-induced p15(INK4b) protein expression. Additionally, N-acetylcysteine (an antioxidant) attenuated high glucose-induced growth arrest and p15(INK4b) protein expression. Thus, high glucose-induced growth arrest is dependent on p15(INK4b) and oxidative stress in endothelial cells. Moreover, high glucose-induced p15(INK4b) protein expression is dependent on ERK1/2 and oxidative stress.

GLUT4 trafficking to the plasma membrane of muscle and fat cells is regulated by insulin. An important component of insulin-regulated GLUT4 distribution is the Akt substrate AS160 rab GTPase-activating protein. Here we show that Rab10 functions as a downstream target of AS160 in the insulin-signaling pathway that regulates GLUT4 translocation in adipocytes. Overexpression of a mutant of Rab10 defective for GTP hydrolysis increased GLUT4 on the surface of basal adipocytes. Rab10 knockdown resulted in an attenuation of insulin-induced GLUT4 redistribution to the plasma membrane and a concomitant 2-fold decrease in GLUT4 exocytosis rate. Re-expression of a wild-type Rab10 restored normal GLUT4 translocation. The basal increase in plasma-membrane GLUT4 due to AS160 knockdown was partially blocked by knocking down Rab10 in the same cells, further indicating that Rab10 is a target of AS160 and a positive regulator of GLUT4 trafficking to the cell surface upon insulin stimulation.

Chemical genetics seeks to identify small molecules that afford functional dissection of cell biological pathways. Previous screens for small molecule inhibitors of exocytic membrane traffic yielded the identification and characterization of several compounds that block traffic from the Golgi to the cell surface as well as transport from the endoplasmic reticulum to the Golgi network [Feng et al. Proc Natl Acad Sci USA 2003;100:6469-6474; Yarrow et al. Comb Chem High Throughput Screen 2003;6:279-286; Feng et al. EMBO Reports 2004: in press]. Here, we screened these inhibitors for potential effects on endocytic membrane traffic. Two structurally related sulfonamides were found to be potent and reversible inhibitors of transferrin-mediated iron uptake. These inhibitors do not block endoplasmic reticulum-to-Golgi transport, but do disrupt Golgi-to-cell surface traffic. The compounds are members of a novel class of sulfonamides that elevate endosomal and lysosomal pH, down-regulate cell surface receptors, and impair recycling of internalized transferrin receptors to the plasma membrane. In vitro experiments revealed that the sulfonamides directly inhibit adenosine triphosphate (ATP) hydrolysis by the V-ATPase and that they also possess a potent proton ionophore activity. While maintenance of organellar pH is known to be a critical factor in both endocytosis and exocytosis, the precise role of acidification, beyond the uncoupling of ligands from their receptors, remains largely unknown. Identification of this novel class of sulfonamide inhibitors provides new chemical tools to better understand the function of organelle pH in membrane traffic and the activity of V-ATPases in particular.

The effects of polymeric IgA1 (pIgA1) and monomeric IgA1 (mIgA1) from patients with IgA nephropathy (IgAN) on the renin-angiotensin system (RAS) and TGF-beta synthesis were examined in cultured human mesangial cells (HMC). Both pIgA1 and mIgA1 induced renin gene expression in HMC, in a dose-dependent manner. Similar findings were observed for TGF-beta gene and protein expression. The values measured in HMC incubated with pIgA1 were significantly higher than those in HMC incubated with equivalent amounts of mIgA1. When similar experiments were performed with the addition of either captopril or losartan, there was a significant increase in the renin gene expression by HMC, whereas the synthesis of TGF-beta was markedly reduced. The TGF-beta signal transduction pathways in HMC were studied by measuring the receptor-regulated Smad proteins (Smad 2 and 3) and common-partner Smad proteins (Smad 4). pIgA1 from patients with IgAN upregulated Smad activity in HMC, and the activity observed in HMC that had been preincubated with pIgA1 was readily suppressed with optimal concentrations of captopril or losartan. The effects of pIgA1 on the RAS were further examined in HMC incubated with IgA isolated from 30 patients with IgAN, 30 healthy subjects, and disease control subjects with other diseases. pIgA1 induction of angiotensin II or TGF-beta synthesis in HMC was significantly greater with preparations from patients with IgAN, compared with healthy or disease control subjects. The findings support a pathogenetic role of pIgA1 in IgAN through upregulation of the RAS and TGF-beta, leading to chronic renal failure with renal fibrosis.

Background: Ketonuria is common in diabetes. The major form of ketone body is beta-hydroxybutyrate (beta-HB), which is metabolized by the proximal tubule. Transforming growth factor beta (TGF-beta) and tubulopathy are important in diabetic nephropathy. Thus, the role of TGF-beta and the downstream Smad3 in beta-HB-induced effects in the human proximal tubule (HK-2 cell) was studied., Methods: Effects of beta-HB (0.1 to 10 mmol/L) on HK-2 cells were determined for: proliferation, cell cycle distribution, collagen production, tubular transdifferentiation [expression of alpha-smooth muscle actin (alpha-SMA) protein], TGF-beta, Smad2/3, p21WAF1, and p27kip1., Results: Beta-HB (0.1 to 10 mmol/L) dose dependently decreased proliferation, arrested the cells in G0/G1 phase of the cell cycle, and increased p21WAF1/p27kip1 protein expression at 48 hours (without affecting p21WAF1/p27kip1 mRNA and transcription). beta-HB (1 mmol/L) increased p21WAF1/p27kip1 protein half-lives. Beta-HB (1 mmol/L) increased TGF-beta transcription at 24 hours and TGF-beta1 mRNA/bioactivity at 48 hours. Beta-HB (1 mmol/L) increased nuclear Smad2/3 protein expression and increased collagen production (without affecting tubular transdifferentiation), which were reversed by Smad7, dominant-negative Smad3, and N-acetylcysteine. Dominant-negative Smad3 reversed beta-HB-induced TGF-beta transcription at 24 hours, and reversed TGF-beta1 bioactivity at 48 hours. Dominant-negative Smad3 reversed beta-HB-induced p21WAF1/p27kip1 protein expression at 48 hours. Finally, N-acetylcysteine, TGF-beta antibody, Smad7, and dominant-negative Smad3 reversed beta-HB (1 mmol/L)-induced growth inhibition at 48 hours., Conclusion: Beta-HB activated Smad 2/3 by oxidative stress. TGF-beta and Smad3 mediate beta-HB-induced cell cycle-dependent growth inhibition while Smad3 mediate beta-HB-induced collagen production and p21WAF1/p27kip1 protein expression in HK-2 cells. Moreover, beta-HB increased p21WAF1/p27kip1 protein expression by increasing p21WAF1/p27kip1 protein stability.