Exosomal lncRNA HCP5 derived from human bone marrow mesenchymal stem cells improves chronic periodontitis by miR-24-3p/HO1/P38/ELK1 pathway.

Objective: The present study aimed to explore the function of human bone marrow mesenchymal stem cells (hBMMSCs)-derived exosomal long noncoding RNA histocompatibility leukocyte antigen complex P5 (HCP5) in the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) to improve chronic periodontitis (CP). Methods: Exosomes were extracted from hBMMSCs. Alizarin red S staining was used to detect mineralised nodules. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to measure HCP5 and miR-24-3p expression. The mRNA and protein levels of alkaline phosphatase (ALP), osteocalcin, osterix, runt-related transcription factor 2, bone morphogenetic protein 2, osteopontin, fibronectin, collagen 1, heme oxygenase 1 (HO1), P38, and ETS transcription factor ELK1 (ELK1) were detected using RT-qPCR and Western blot. Enzyme-linked immunosorbent assay (ELISA) kits were used to determine the HO1 and carbon monoxide concentrations. Heme, biliverdin, and Fe2+ levels were determined using detection kits. Micro-computed tomography, hematoxylin and eosin staining, ALP staining, tartrate-resistant acid phosphatase staining, ELISA, and RT-qPCR were conducted to evaluate the effect of HCP5 on CP mice. Dual luciferase, RNA immunoprecipitation, and RNA pulldown experiments were performed to identify the interactions among HCP5, miR-24-3p, and HO1. Results: The osteogenic ability of hPDLSCs significantly increased when co-cultured with hBMMSCs or hBMMSCs exosomes. Overexpression of HCP5 and HO1 in hBMMSCs exosomes promoted the osteogenic differentiation of hPDLSCs, and knockdown of HCP5 repressed the osteogenic differentiation of hPDLSCs. HCP5 knockdown enhanced the inflammatory response and repressed osteogenesis in CP mice. MiR-24-3p overexpression diminished the stimulatory effect of HCP5 on the osteogenic ability of hPDLSCs. Mechanistically, HCP5 acted as a sponge for miR-24-3p and regulated HO1 expression, and HO1 activated the P38/ELK1 pathway. Conclusion: HBMMSCs-derived exosomal HCP5 promotes the osteogenic differentiation of hPDLSCs and alleviates CP by regulating the miR-24-3p/HO1/P38/ELK1 signalling pathway.

Functional characterization of the InR/PI3K/AKT signaling pathway in female reproduction of the predatory bug Cyrtorhinus lividipennis (Hemiptera: Miridae).

The insulin signaling (IIS) pathway plays a key role in the regulation of various physiological functions in animals. However, the involvement of IIS pathway in the reproduction of natural enemy insects remains enigmatic. Here, 3 key genes (named ClInR, ClPI3K, and ClAKT) related to IIS pathway were cloned from Cyrtorhinus lividipennis (Reuter) (Hemiptera: Miridae), an important natural enemy in the rice ecosystem. These 3 proteins had the typical features of corresponding protein families and shared high similarity with their respective homologs from the Hemipteran species. The ClInR, ClPI3K, and ClAKT were highly expressed in the adult stage. Tissue distribution analysis revealed that ClInR, ClPI3K, and ClAKT were highly expressed in the midgut and ovary of adults. Silencing of ClInR, ClPI3K, and ClAKT caused 92.1%, 72.1%, and 57.8% reduction in the expression of ClVg, respectively. Depletion of these 3 genes impaired vitellogenin synthesis and ovary development. Moreover, the fecundity in the dsInR, dsPI3K, and dsAKT injected females were 53.9%, 50.8%, and 48.5% lower than the control treatment, respectively. These results indicated that ClInR, ClPI3K, and ClAKT are of great importance for the reproduction of C. lividipennis. Our results advance the knowledge about the molecular mechanism of reproduction regulation in natural enemy insects.

Ferrostatin-1 post-treatment attenuates acute kidney injury in mice by inhibiting ferritin production and regulating iron uptake-related proteins.

Background: Acute kidney injury (AKI) is a common and serious medical condition with high morbidity and mortality. Recent research has highlighted ferroptosis, a novel form of programmed cell death, as a potential therapeutic target in mitigating renal tubular injury in AKI. Ferrostatin-1, a specific ferroptosis inhibitor, has been demonstrated to prevent renal injury through ferroptosis inhibition. Methods: Utilizing a murine AKI model, we investigated the effects of Ferrostatin-1 by administering it post-injury. Through high-throughput sequencing and pathological analysis, we focused on the critical role of ferroptosis-related pathways in the treatment. Results: Ferrostatin-1 post-conditioning effectively mitigated oxidative damage and reduced iron content associated with AKI. Additionally, critical ferroptosis-related proteins, such as GPX4, SLC7A11, NRF2, and FTH1, exhibited increased expression levels. In vitro, Ferrostatin-1 treatment of HK-2 cells significantly diminished lipid peroxidation and iron accumulation. Furthermore, Ferrostatin-1 was found to downregulate the PI3K signalling pathway. Conclusion: Ferrostatin-1 acted as a potential ferroptosis inhibitor with the capacity to enhance antioxidant defences. This study suggests that Ferrostatin-1 could serve as a promising novel strategy for improving the treatment of AKI and promoting recovery from the condition.

Adaptive prescribed settling time periodic event-triggered control for uncertain robotic manipulators with state constraints.

In this paper, an adaptive prescribed settling time periodic event-triggered control (APST-PETC) is investigated for uncertain robotic manipulators with state constraints. In order to economize network bandwidth occupancy and reduce computational burden, a periodic event-triggered control (PETC) strategy is proposed to reduce the update frequency of the control signal and avoid unnecessary continuous monitoring. Besides, considering that the maneuverable space of the actual robotic manipulators is often limited, the barrier Lyapunov function (BLF) is applied to deal with the influence of the constraint characteristics on the robotic manipulators. Further, based on the one-to-one nonlinear mapping function of the system tracking error, an adaptive prescribed settling time control (APSTC) is designed to ensure that the system tracking error reaches the predetermined precision residual set within the prescribed settling time. Finally, theoretical analysis and comparative experiments are given to verify its feasibility.

[Corrigendum] Epithelial mesenchymal transition induced by the CXCL9/CXCR3 axis through AKT activation promotes invasion and metastasis in tongue squamous cell carcinoma.

Subsequently to the publication of this paper, an interested reader drew to the authors' attention that two pairs of data panels containing strikingly similar data were featured in Fig. 4A and B. The authors have re­examined their data and realized that Fig. 4 was assembled incorrectly. The revised version of Fig. 4, containing the correct data for Fig. 4A and B, is shown on the next page. The authors regret the errors that were made in the preparation of the published figure, and confirm that these errors did not seriously affect the conclusions reported in the paper. The authors are grateful to the editor of Oncology Reports for allowing them the opportunity to publish a Corrigendum, and all the authors agree to this Corrigendum. Furthermore, they apologise to the readership for any inconvenience caused. [the original article was published in Oncology Reports 39: 1356-1368, 2018; DOI: 10.3892/or.2017.6169].

The pH-triggered polyglutamate brush co-delivery of MDR1 and survivin-targeting siRNAs efficiently overcomes multi-drug resistance of NSCLC.

Multi-drug resistance (MDR) is one of the major challenges in the successful chemotherapy of non-small cell lung cancer (NSCLC). Although RNA interference (RNAi) has been widely used to silence resistance-related genes, the effect remains unsatisfactory. In this study, we attempted to overcome MDR of NSCLC by simultaneously interfering with two RNAs that have different functions. A new pH-triggered polyglutamate brush polymer dimethylmaleic anhydride-poly(ethyleneglycol) monomethyl ether-b-polyglutamate-g-spermine (DMA-mPEG-b-PG-g-spermine, DPPGS) was designed and synthesized. The DPPGS/small interfering RNA (siRNA) complex nanoparticles (DPPGSN) were prepared. The results demonstrated that DPPGSN could be transformed from a negatively charged form into a positively charged form in the slightly acidic tumor extracellular environment. The siRNA targeting MDR1 mRNA (siMDR1) and siRNA targeting survivin mRNA (siSurvivin) could be efficiently co-delivered by DPPGS to simultaneously interfere with two genes (p < 0.01). Furthermore, DPPGS co-delivery of siMDR1 and siSurvivin lowered the IC50 value of cisplatin (DDP) in A549/DDP (p < 0.01) cells and increased the apoptosis rate of the cells (p < 0.01). Therefore, co-delivery of siMDR1 and siSurvivin using DPPGS would be a promising approach for overcoming MDR of NSCLC.

Epithelial mesenchymal transition induced by the CXCL9/CXCR3 axis through AKT activation promotes invasion and metastasis in tongue squamous cell carcinoma.

The present study aimed to assess the induction of epithelial-mesenchymal transition (EMT), invasion, and metastasis by the chemokine CXCL9/receptor CXCR3 axis in tongue squamous cell carcinoma (TSCC), unveiling the underlying mechanisms and providing new insights into the prevention and treatment of oral cancer metastasis. The expression levels of CXCL9 and CXCR3 in TSCC tissue specimens were determined by immunohistochemistry, assessing differences between samples with cervical lymph node metastasis and those without. Moreover, protein expression or activity in the TSCC Cal-27 cell line was controlled by neutralizing antibodies, gene transfection, or knock-out. Then, alterations of cell proliferation, migration, invasion, and the cytoskeleton were analyzed by CCK-8, cell scratch, Transwell, and cyto-skeleton staining assays, respectively. Alterations of EMT markers (E-cadherin and vimentin) in Cal-27 cells were detected by immunofluorescence and western blotting. In addition, western blotting was utilized to detect protein expression levels of Akt2, p-Akt2, eIF4E and p-eIF4E, and to explore the regulatory roles and mechanisms of the CXCL9/CXCR3 axis in invasion and metastasis. Significantly increased expression levels of CXCL9 and CXCR3 were detected in tissue specimens with lymph node metastasis compared with those without (P<0.01). Overexpression of CXCL9/CXCR3 in Cal-27 cells resulted in cytoskeleton alterations, decreased E-cadherin expression, increased vimentin levels, enhanced migration and invasion (P<0.05), and increased phosphorylated Akt2 and eIF4E levels (P<0.05). These results revealed that in TSCC, the CXCL9/CXCR3 axis could activate the Akt signaling pathway, with EMT and cytoskeleton rearrangement, promoting invasion and metastasis.

Epithelial cell-derived micro RNA-146a generates interleukin-10-producing monocytes to inhibit nasal allergy.

The aberrant immunity plays an important role in the pathogenesis of allergic diseases. Micro RNAs (miR) are involved in regulating the immunity in the body. This study aims to test a hypothesis that miR-146a induces the expression of interleukin (IL)-10 in monocytes (Mos). In this study, the levels of miR-146a were determined by real time RT-PCR. The IL-10(+) Mos were evaluated by flow cytometry. The miR-146a-laden exosomes were generated with RPMI2650 cells (an airway epithelial cell line). An allergic rhinitis mouse model was developed. The results showed that nasal epithelial cells expressed miR-146a, which was markedly lower in the nasal epithelial cells of patients with nasal allergy than that in healthy controls. Exposure to T helper (Th)2 cytokines suppressed the levels of miR-146a in the nasal epithelial cells. The nasal epithelial cell-derived miR-146a up regulated the expression of IL-10 in Mos. The inducible IL-10(+) Mos showed an immune suppressor effect on the activities of CD4(+) effector T cells and the Th2 polarization in a mouse model of allergic rhinitis. In summary, nasal epithelial cells express miR-146a, the latter is capable of inducing IL-10 expression in Mos, which suppress allergic reactions in the mouse nasal mucosa.

Identification of proteins interacting with protein kinase C-δ in hyperthermia-induced apoptosis and thermotolerance of Tca8113 cells.

The purpose of the present study was to investigate the differential proteins that interact with protein kinase C­Î´ (PKC­Î´) in hyperthermia­induced apoptosis as well as thermotolerance in Tca8113 cells, and furthermore, to investigate the mechanisms of these processes in tumor cells. Activation of PKC­Î´ was previously indicated to be involved in the heat sensitivity and thermal resistance of tongue squamous carcinoma cells. Tca8113 cell apoptosis was induced by incubation at 43˚C for 80 min and the thermotolerant Tca8113 cells (TT­Tca8113) were generated through a gradient temperature­elevating method. The apoptotic rate of the cells was determined by flow cytometry, while cleavage and activation of PKC­Î´ were analyzed by western blot analysis. The proteins that interacted with PKC­Î´ in the Tca8113 and TT­Tca8113 cells were identified by co­immunoprecipitation coupled with mass spectrometry. Co­immunoprecipitation analysis followed by electrospray ionization mass spectrometric analysis were utilized to identify the pro­ and anti­apoptotic proteins that interacted with PKC­Î´. Significant cell apoptosis was observed in Tca8113 cells following hyperthermia, and the apoptotic rate was significantly higher than that in the control group (P<0.05). Marked PKC­Î´ cleavage fragmentation was also identified. By contrast, the apoptotic rate of the TT­Tca8113 cells was not significantly increased following hyperthermia and no PKC­Î´ cleavage fragmentation was observed. Among the proteins interacting with PKC­Î´, 39 were found to be involved in the promotion of apoptosis and 16 in the inhibition of apoptosis of Tca8113 cells; these proteins were known to be involved in the regulation of cell proliferation, apoptosis, transcription and intracellular protein transport. The results of the present study provided evidence that PKC­Î´ is a crucial factor in the heat sensitivity and thermal resistance of tongue squamous carcinoma cells and elucidated the underlying molecular basis, which may aid in the improvement of hyperthermic cancer treatments.

Synthesis of Deuterated Benzopyran Derivatives as Selective COX-2 Inhibitors with Improved Pharmacokinetic Properties.

We designed a series of specifically deuterated benzopyran analogues as new COX-2 inhibitors with the aim of improving their pharmacokinetic properties. As expected, the deuterated compounds retained potency and selectivity for COX-2. The new molecules possess improved pharmacokinetic profiles in rats compared to their nondeuterated congeners. Most importantly, the new compounds showed pharmacodynamic efficacy in several murine models of inflammation and pain. The benzopyran derivatives were separated into their enantiomers, and the activity was found to reside with the S-isomers. To streamline the synthesis of the desired S-isomers, an organocatalytic asymmetric domino oxa-Michael/aldol condensation reaction was developed for their preparation.

Identification of GZD824 as an orally bioavailable inhibitor that targets phosphorylated and nonphosphorylated breakpoint cluster region-Abelson (Bcr-Abl) kinase and overcomes clinically acquired mutation-induced resistance against imatinib.

Bcr-Abl(T315I) mutation-induced imatinib resistance remains a major challenge for clinical management of chronic myelogenous leukemia (CML). Herein, we report GZD824 (10a) as a novel orally bioavailable inhibitor against a broad spectrum of Bcr-Abl mutants including T315I. It tightly bound to Bcr-Abl(WT) and Bcr-Abl(T315I) with K(d) values of 0.32 and 0.71 nM, respectively, and strongly inhibited the kinase functions with nanomolar IC(50) values. The compound potently suppressed proliferation of Bcr-Abl-positive K562 and Ku812 human CML cells with IC(50) values of 0.2 and 0.13 nM, respectively. It also displayed good oral bioavailability (48.7%), a reasonable half-life (10.6 h), and promising in vivo antitumor efficacy. It induced tumor regression in mouse xenograft tumor models driven by Bcr-Abl(WT) or the mutants and significantly improved the survival of mice bearing an allograft leukemia model with Ba/F3 cells harboring Bcr-Abl(T315I). GZD824 represents a promising lead candidate for development of Bcr-Abl inhibitors to overcome acquired imatinib resistance.

Expression of adrenomedullin in human ovaries, ovarian sex cord-stromal tumors and cultured granulosa-luteal cells.

The aim of the present study was to characterise the expression pattern of the multifunctional vasoactive peptide adrenomedullin (ADM) in human ovarian tumors, and to find hormonal regulators of ADM expression in human ovaries. The expression of ADM messenger RNA (mRNA) was higher in granulosa cell tumors than in fibrothecomas and normal ovaries, as analysed by Northern blots. In normal ovaries, ADM immunoreactivity was localised in both granulosa and thecal cells. Eight of the 90 granulosa cell tumors (9%) showed moderate and 53 (59%) weak ADM immunoreactivity, whereas 27% (11/41) of the fibrothecomas displayed weak ADM staining. FSH, protein kinase A activator (Bu)(2)cAMP, prostaglandin E(2) (PGE(2)), activin A and the broad protein kinase regulator staurosporine decreased ADM mRNA accumulation in cultured granulosa-luteal cells time- and dose-dependently. FSH, (Bu)(2)cAMP and PGE(2) increased progesterone secretion and the accumulation of the steroidogenic acute regulatory protein mRNA in these cells. In conclusion, ADM is expressed in normal human ovaries and sex cord-stromal tumors, particularly in those of granulosa cell origin. FSH, PGE(2,) (Bu)(2)cAMP and activin A suppress ADM gene expression in granulosa-luteal cells. Expression of ADM in human ovaries and its hormonal regulation in granulosa cells suggests a paracrine role for ADM in ovarian function.

Inhibition of DNA methylation increases follistatin expression and secretion in the human adrenocortical cell line NCI-H295R.

Activin affects adrenocortical steroidogenesis and increases apoptosis, while follistatin (FS) acts as an activin antagonist by binding to activin, preventing attachment to its receptors. The regulation of FS expression in the adrenal cortex is poorly understood. Adrenocortical tumors often display aberrant methylation. In the present study, we investigated the effect of DNA methylation on FS mRNA expression and peptide secretion in adrenocortical cells. We treated human NCI-H295R adrenocortical cells with the methylation inhibitor 5-Aza-2'deoxycytidine (Azad; 0.1-100 microM for 1, 4 or 7 days) and measured FS mRNA expression by Northern blot and quantitative real time RT-PCR analyses as well as FS secretion by specific ELISA. Methylation-specific PCR showed decreased methylation in the FS promoter region after Azad treatment. A significant (P < 0.05) time- and dose-dependent increase in FS mRNA expression (up to 4.6-fold) and peptide secretion (up to 17.1-fold) was detected after Azad treatment. We conclude that FS gene expression and peptide secretion in NCI-H295R adrenocortical cells are regulated by DNA methylation. Thus, variable methylation in different adrenocortical tumors may influence activin bioactivity and its consequences in steroidogenesis and cell proliferation/apoptosis.

Transcription factors GATA-6, SF-1, and cell proliferation in human adrenocortical tumors.

Transcription factor GATA-6 is expressed in fetal and adult human adrenal cortex and has been suggested to have a role in adrenal androgen synthesis. In other tissues GATA-6 has been linked to the cell cycle regulation and the dedifferentiation of carcinoma cells. GATA-6 has been shown to be downregulated in mouse adrenocortical tumors, but has not been studied in human adrenocortical tumors in detail. We have now analyzed GATA-6 expression in 20 human adrenocortical adenomas and 16 carcinomas using Northern blot analysis and immunohistochemistry. GATA-6 mRNA and protein expression was remarkably diminished in adrenocortical carcinomas as compared to normal adrenal cortex and adenomas (p<0.05). In opposite to other tumor types GATA-6 expression was, however, high in virilizing carcinomas. Steroidogenic factor 1 (SF-1) has been functionally linked to GATA-6, and the expression of these two factors correlated in the adrenal tumors. Furthermore, GATA-6 immunoreactivity was linked to P450c17 expression. In contrast to GATA-6, we found upregulated cyclin-dependent kinase inhibitor p21 and proliferation marker Ki67 in adrenocortical carcinomas indicating that GATA-6 is not linked to cell proliferation in human adrenal tumors. Taken together, the present and earlier results link GATA-6 to adrenocortical steroidogenesis and to the benign adrenocortical phenotype.

Expression of betaglycan, an inhibin coreceptor, in normal human ovaries and ovarian sex cord-stromal tumors and its regulation in cultured human granulosa-luteal cells.

Activins and inhibins are often antagonistic in the regulation of ovarian function. TGFbeta type III receptor, betaglycan, has been identified as a coreceptor to enhance the binding of inhibins to activin type II receptor and thus to prevent the binding of activins to their receptor. In this study we characterized the expression and regulation pattern of betaglycan gene in normal ovaries and sex cord-stromal tumors and in cultured human granulosa-luteal cells from women undergoing in vitro fertilization. Expression of betaglycan mRNA was detected by RT-PCR or Northern blotting in normal ovarian granulosa, thecal, and stroma cells as well as in granulosa-luteal cells. Immunohistochemical analysis revealed positive staining for betaglycan in antral and preovulatory follicular granulosa and thecal cells and in corpora lutea of normal ovaries. Furthermore, betaglycan expression was detected in the vast majority of granulosa cell tumors, thecomas, and fibromas, with weaker staining in granulosa cell tumors compared with fibrothecomas. In cultured granulosa-luteal cells, FSH and LH treatment increased dose-dependently the accumulation of betaglycan mRNA, as did the protein kinase A activator dibutyryl cAMP and the protein kinase C inhibitor staurosporine. In contrast, the protein kinase C activator 12-O-tetradecanoyl phorbol 13-acetate had no significant effect on betaglycan mRNA levels. Treatment with prostaglandin E(2) and with its receptor EP2 subtype agonist butaprost increased betaglycan mRNA accumulation and progesterone secretion dose- and time-dependently. In summary, betaglycan gene is expressed in normal human ovarian steroidogenic cells and sex cord-stromal ovarian tumors. The accumulation of its mRNA in cultured granulosa-luteal cells is up-regulated by gonadotropins and prostaglandin E(2), probably via the protein kinase A pathway. The specific expression and regulation pattern of betaglycan gene may be related to the functional antagonism of inhibins to activin signal transduction in human ovaries.

VEGF in 105 pheochromocytomas: enhanced expression correlates with malignant outcome.

Pheochromocytomas are rare sympathoadrenal tumors that are highly vascular. Their malignancy is extremely difficult to estimate on the basis of histopathological features. Vascular endothelial growth factor (VEGF) is one of the most important angiogenic factors involved in both tumor growth and metastasis. In our search for new prognostic markers, we investigated the expression of VEGF in normal adrenal gland, in 105 primary pheochromocytomas, and in 6 metastases by using immunohistochemistry and Northern blot analysis. We also calculated the microvessel density of these tumors by staining the endothelial cells with monoclonal CD34 antibody. VEGF messenger ribonucleic acid was found in all pheochromocytomas studied. Immunohistochemically, VEGF was not found in normal adrenal medullary cells. Interestingly, all malignant pheochromocytomas (n=8), regardless of their primary location, had strong or moderate VEGF immunoreactivity, while most benign adrenal pheochromocytomas (26 of 37, 70.3%) were either negative or only weakly positive. The staining was heterogenous in extraadrenal pheochromocytomas as well as in a group of tumors that had histologically suspicious features but had not metastasized, here called borderline tumors (n=29). The microvessel density varied greatly in all of the tumor groups, and no statistical difference was found between these groups. Here we report moderate to strong VEGF expression in malignant pheochromocytomas, and negative or weak expression in benign adrenal pheochromocytomas. Normal medullary cells are immunohistochemically negative. Thus, low VEGF expression in pheochromocytomas favors a benign diagnosis.

Expression of activin/inhibin receptor and binding protein genes and regulation of activin/inhibin peptide secretion in human adrenocortical cells.

Activins and inhibins are glycoprotein hormones produced mainly in gonads but also in other organs. They are believed to be important para/autocrine regulators of various cell functions. We investigated activin/inhibin receptor and binding protein gene expression and the regulation of activin/inhibin secretion in human adrenal cells. RT-PCR revealed inhibin/activin alpha-, betaA/B-subunit, follistatin, activin type I/II receptor, and inhibin receptor (betaglycan and inhibin-binding protein) mRNA expression in fetal and adult adrenals and cultured adrenocortical cells. Cultured cells secreted activin A and inhibin A/B as determined by specific ELISAs. ACTH stimulated inhibin A/B secretion in fetal (1.8- and 1.8-fold of control, respectively) and in adult cells (3.4- and 1.7-fold of control, respectively) without significant effect on activin A. 8-bromoadenosine cAMP (protein kinase A activator) increased activin A and inhibin A/B secretion in the human adrenocortical NCI-H295R cell line (32-, 17-, and 3-fold of control, respectively). 12-O-tetradecanoyl phorbol-13-acetate (protein kinase C activator) stimulated both activin A and inhibin A secretion (764- and 32-fold of control, respectively), and activin treatment increased inhibin B secretion in these cells (25-fold of control). In conclusion, human adrenocortical cells produce dimeric activins and inhibins. ACTH stimulates inhibin secretion and decreases activin/inhibin secretion ratio, probably via the protein kinase A signal transduction pathway. This, together with the adrenocortical activin/ inhibin receptor and binding protein expression, suggests a physiological role for activins and inhibins in the human adrenal gland.

Association of H19 promoter methylation with the expression of H19 and IGF-II genes in adrenocortical tumors.

Low H19 and abundant IGF-II expression may have a role in the development of adrenocortical carcinomas. In the mouse, the H19 promoter area has been found to be methylated when transcription of the H19 gene is silent and unmethylated when it is active. We used PCR-based methylation analysis and bisulfite genomic sequencing to study the cytosine methylation status of the H19 promoter region in 16 normal adrenals and 30 pathological adrenocortical samples. PCR-based analysis showed higher methylation status at three HpaII-cutting CpG sites of the H19 promoter in adrenocortical carcinomas and in a virilizing adenoma than in their adjacent normal adrenal tissues. Bisulfite genomic sequencing revealed a significantly higher mean degree of methylation at each of 12 CpG sites of the H19 promoter in adrenocortical carcinomas than in normal adrenals (P < 0.01 for all sites) or adrenocortical adenomas (P < 0.01, except P < 0.05 for site 12 and P > 0.05 for site 11). The mean methylation degree of the 12 CpG sites was significantly higher in the adrenocortical carcinomas (mean +/- SE, 76 +/- 7%) than in normal adrenals (41 +/- 2%) or adrenocortical adenomas (45 +/- 3%; both P < 0.005). RNA analysis indicated that the adrenocortical carcinomas expressed less H19 but more IGF-II RNAs than normal adrenal tissues did. The mean methylation degree of the 12 H19 promoter CpG sites correlated negatively with H19 RNA levels (r = -0.550; P < 0.01), but positively with IGF-II mRNA levels (r = 0.805; P < 0.001). In the adrenocortical carcinoma cell line NCI-H295R, abundant IGF-II, but minimal H19, RNA expression was detected by Northern blotting. Treatment with a cytosine methylation inhibitor, 5-aza-2'-deoxycytidine, increased H19 RNA expression, whereas it decreased IGF-II mRNA accumulation dose- and time-dependently (both P < 0.005) and reduced cell proliferation to 10% in 7 d. Our results suggest that altered DNA methylation of the H19 promoter is involved in the abnormal expression of both H19 and IGF-II genes in human adrenocortical carcinomas.

Gonadotrophins inhibit the expression of insulin-like growth factor binding protein-related protein-2 mRNA in cultured human granulosa-luteal cells.

Insulin-like growth factors (IGF) and IGF-binding proteins (IGFBP) have been shown to be involved in ovarian follicular growth/development and steroidogenesis. Recently, a number of low-affinity IGFBP-related proteins (IGFBP-rP) have been characterized. In this study, we investigated the expression of the gene for IGFBP-rP2 (also known as connective tissue growth factor, CTGF) in human granulosa cells in vitro and in vivo. Northern blot analysis demonstrated that IGFBP-rP2 mRNA is expressed in cultured human granulosa-luteal cells obtained from women undergoing an IVF programme. Accumulation of IGFBP-rP2 mRNA was dose-dependently down-regulated by FSH and LH after 24 h treatment (both P < 0.05) in cultured granulosa-luteal cells. The inhibitory effects of gonadotrophins were mimicked by treatment with the protein kinase A activator, (Bu)(2)cAMP. Protein kinase C inhibitor staurosporine reduced, whereas protein kinase C activator TPA (12-O-tetradecanoyl phorbol 13-acetate) increased, IGFBP-rP2 mRNA accumulation. These results suggest that the inhibitory effects of gonadotrophins on IGFBP-rP2 gene expression may involve signal transduction via both protein kinase A and C pathways. Immunohistochemical analysis revealed positive staining for IGFBP-rP2 in the granulosa and theca cells of normal human ovarian follicles. Corpus luteum and ovarian surface epithelial cells were also positively stained. Modulation of IGFBP-rP2 expression by gonadotrophic hormones may have a role in ovarian follicular development and in the ovulatory process.