Effects of natural 24-epibrassinolide on inducing apoptosis and restricting metabolism in hepatocarcinoma cells.

BACKGROUND: 24-epibrassinolide (EBR) is a ubiquitous steroidal phytohormone with anticancer activity. Yet the cytotoxic effects and mechanism of EBR on hepatocarcinoma (HCC) cells remain elusive. METHODS: Cell counting kit-8 (CCK-8) assay was performed to evaluate cell viability. Real-time cell analysis (RTCA) technology and colony formation assays were used to evaluate cell proliferation. The apoptosis ratio was measured by flow cytometry. Seahorse XFe96 was applied to detect the effects of EBR on cellular bioenergetics. RNA-seq analysis was performed to investigate differences in gene expression profiles. Western blot and qRT-PCR were used to detect the changes in target molecules. RESULTS: EBR induced apoptosis and caused energy restriction in HCC, both of which were related to insulin-like growth factor-binding protein 1 (IGFBP1). EBR rapidly and massively induced IGBFP1, part of which was transcribed by activating transcription factor-4 (ATF4). The accumulation of secreted and cellular IGFBP1 had different important roles, in which secreted IGFBP1 affected cell energy metabolism by inhibiting the phosphorylation of Akt, while intracellular IGFBP1 acted as a pro-survival factor to resist apoptosis. Interestingly, the extracellular signal-regulated kinase (ERK) inhibitor SCH772984 and MAP/ERK kinase (MEK) inhibitor PD98059 not only attenuated the EBR-induced IGFBP1 expression but also the basal expression of IGFBP1. Thus, the treatment of cells with these inhibitors further enhances the cytotoxicity of EBR. CONCLUSION: Taken together, these findings suggested that EBR can be considered as a potential therapeutic compound for HCC due to its pro-apoptosis, restriction of energy metabolism, and other anti-cancer properties. Meanwhile, the high expression of IGFBP1 induced by EBR in HCC contributes to our understanding of the role of IGFBP1 in drug resistance.

Long non-coding RNA IGFBP7-AS1 accelerates the odontogenic differentiation of stem cells from human exfoliated deciduous teeth by regulating IGFBP7 expression.

Stem cells from human exfoliated deciduous teeth (SHED) are attractive seed cells for dental tissue engineering. We identified the effect of the long noncoding RNA insulin-like growth factor-binding protein 7 antisense RNA 1 (lncRNA IGFBP7-AS1) in vivo and its underlying mechanism during SHED odontogenic differentiation. IGFBP7-AS1 and insulin-like growth factor-binding protein 7 (IGFBP7) were overexpressed using lentiviruses. IGFBP7 expression was knocked down with small interfering RNA. The effect of IGFBP7-AS1 in vivo was confirmed by animal experiments. The effect of IGFBP7 on SHED odontogenic differentiation was assessed with alkaline phosphatase staining, alizarin red S staining, quantitative reverse transcription-PCR, and western blotting. The relationship between IGFBP7-AS1 and IGFBP7 was confirmed by quantitative reverse transcription-PCR and western blotting. IGFBP7-AS1 promoted SHED odontogenesis in vivo, and regulated the expression of the coding gene IGFBP7 positively. Inhibiting IGFBP7 led to suppress SHED odontogenic differentiation while IGFBP7 overexpression had the opposite effect. IGFBP7-AS1 enhanced the stability of IGFBP7. IGFBP7-AS1 promoted SHED odontogenic differentiation in vivo. The underlying mechanism may involve the enhancement of IGFBP7 stability. This may provide novel potential targets for dental tissue engineering.

Antimicrobial peptide derived from insulin-like growth factor-binding protein 5 improves diabetic wound healing.

Impaired keratinocyte functions are major factors that are responsible for delayed diabetic wound healing. In addition to its antimicrobial activity, the antimicrobial peptide derived from insulin-like growth factor-binding protein 5 (AMP-IBP5) activates mast cells and promotes keratinocyte and fibroblast proliferation and migration. However, its effects on diabetic wound healing remain unclear. Human keratinocytes were cultured in normal or high glucose milieus. The production of angiogenic growth factor and cell proliferation and migration were evaluated. Wounds in normal and streptozotocin-induced diabetic mice were monitored and histologically examined. We found that AMP-IBP5 rescued the high glucose-induced attenuation of proliferation and migration as well as the production of angiogenin and vascular endothelial growth factors in keratinocytes. The AMP-IBP5-induced activity was mediated by the epidermal growth factor receptor, signal transducer and activator of transcription 1 and 3, and mitogen-activated protein kinase pathways, as indicated by the inhibitory effects of pathway-specific inhibitors. In vivo, AMP-IBP5 markedly accelerated wound healing, increased the expression of angiogenic factors and promoted vessel formation in both normal and diabetic mice. Overall, the finding that AMP-IBP5 accelerated diabetic wound healing by protecting against glucotoxicity and promoting angiogenesis suggests that AMP-IBP5 might be a potential therapeutic target for treating chronic diabetic wounds.

N6-methyladenosine reader YTH N6-methyladenosine RNA binding protein 3 or insulin like growth factor 2 mRNA binding protein 2 knockdown protects human bronchial epithelial cells from hypoxia/reoxygenation injury by inactivating p38 MAPK, AKT, ERK1/2, and NF-κB pathways.

Lung ischemia/reperfusion (I/R) injury (LIRI) is a common complication after lung transplantation, embolism, and trauma. N6-methyladenosine (m6A) methylation modification is implicated in the pathogenesis of I/R injury. However, there are no or few reports of m6A-related regulators in LIRI till now. In this text, dysregulated genes in lung tissues of LIRI rats versus the sham group were identified by RNA sequencing (RNA-seq). RNA-seq outcomes revealed that only YTH N6-methyladenosine RNA binding protein 3 (YTHDF3) and insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) were differentially expressed in the LIRI versus sham group among 20 m6A-related regulators. Next, the functions and molecular mechanisms of YTHDF3 and IGF2BP2 in LIRI were investigated in a hypoxia/reoxygenation-induced BEAS-2B cell injury model in vitro. Results showed that YTHDF3 or IGF2BP2 knockdown attenuated hypoxia/reoxygenation-mediated inhibitory effects on cell survival and cell cycle progression and inhibited hypoxia/reoxygenation-induced cell apoptosis and pro-inflammatory cytokine secretion in BEAS-2B cells. Genes that could be directly regulated by YTHDF3 or IGF2BP2 were identified based on prior experimental data and bioinformatics analysis. Moreover, multiple potential downstream pathways of YTHDF3 and IGF2BP2 were identified by the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analysis of the above-mentioned genes. Among these potential pathways, we demonstrated that YTHDF3 or IGF2BP2 knockdown inhibited hypoxia/reoxygenation-activated p38, ERK1/2, AKT, and NF-κB pathways in BEAS-2B cells. In conclusion, YTHDF3 or IGF2BP2 knockdown weakened hypoxia/reoxygenation-induced human lung bronchial epithelial cell injury by inactivating p38, AKT, ERK1/2, and NF-κB pathways.

IGF-1 treatment during in vitro maturation improves developmental potential of ovine oocytes through the regulation of PI3K/Akt and apoptosis signaling.

This study aimed to assess the effect of the insulin-like grow factor 1 (IGF-1) treatment during in vitro maturation on the gene expression and developmental ability of ovine oocytes. Ovine cumulus-oocyte complexes (COC) were matured in vitro without (control) or with the supplementation of IGF-1 (100 ng/ml) and then subjected to in vitro fertilization and culture. The rate of oocyte maturation and embryo development was recorded and expression of the selected genes (involved in the PI3K/Akt and apoptosis signaling) was assessed in the matured oocytes. The IGF-1 treatment significantly (p < .05) improved the oocyte maturation rate (%) as compared to the control (81.5 ± 2.40 vs. 73.6 ± 0.94). Similarly, as compared to the control, the IGF-1 treatment significantly (p < .05) improved the rate (%) of cleavage (54.7 ± 1.58 vs. 67.2 ± 3.65) and the formation of 4-8 cell embryos (30.7 ± 2.89 vs. 44.1 ± 4.01) and morula (20.7 ± 2.08 vs. 32.8 ± 2.78). The IGF-1 treatment significantly (p < .05) upregulated the expression of IGF1R, PI3KR1, AKT1 and BCL2 and downregulated the expression of GSK3ß, FOXO3 and CASP9 in the matured oocytes. In conclusion, the IGF-1 treatment significantly improved the developmental competence of ovine oocytes through the regulation of the PI3K/Akt and apoptosis signaling.

The Relationship between Diabetes Mellitus Type II and Intervertebral Disc Degeneration in Diabetic Rodent Models: A Systematic and Comprehensive Review.

The number of diabetic patients grows constantly worldwide. Many patients suffer simultaneously from diabetes mellitus type 2 (T2DM) and intervertebral disc disease (IVDD), suggesting a strong link between T2DM and IVDD. T2DM rodent models provide versatile tools to study this interrelation. We hypothesized that the previously achieved studies in rodents approved it. Performing a search in the publicly available electronic databases according to our inclusion (e.g., experimental study with clearly outlined methods investigating IVDD in diabetic rodent models) and exclusion (e.g., non-experimental) criteria, we included 23 studies from 1992 to 2020 analyzing different aspects of IVDD in diabetic rodents, such as on pathogenesis (e.g., effects of hyperglycemia on IVD cells, sirtuin (SIRT)1/p53 axis in the interrelation between T2DM and IVDD), risk factors (e.g., high content of advanced glycation end-products (AGEs) in modern diets), therapeutical approaches (e.g., insulin-like growth factor (IGF-I)), and prophylaxis. Regarding their quality, 12 studies were classified as high, six as moderate, and five as low. One strong, 18 moderate, and three mild evidences of the link between DM and IVDD in rodents were found, while only one study has not approved this link. We concluded that T2DM has a devastating effect on IVD, particularly in advanced cases, which needs to be further evaluated.

IGFs Potentiate TAC3-induced SLα Expression via Upregulation of TACR3 Expression in Grass Carp Pituitary Cells.

In mammals, the tachykinin 3 (TAC3)/tachykinin receptor 3 (TACR3) systems have been confirmed to play an important role in the regulation of puberty onset. Using grass carp pituitary cells as the model, our recent study found that the TAC3 gene products could significantly induce somatolactin α (SLα) synthesis and secretion via TACR3 activation. In the present study, we seek to examine if pituitary TACR3 can serve as a regulatory target and contribute to TAC3 interactions with other SLα regulators. Firstly, grass carp TACR3 was cloned and tissue distribution showed that it could be highly detected in grass carp pituitary. Using HEK293 cells as the model, functional expression also revealed that grass carp TACR3 exhibited ligand binding selectivity and post-receptor signaling highly comparable to its mammalian counterpart. Using grass carp pituitary cells as the model, TACR3 mRNA expression could be stimulated by insulin-like growth factor (IGF)-I and -II via the IGF-I receptor coupled to phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt) and mitogen-activated protein kinase (MAPK) pathways. Interestingly, IGF-I/-II cotreatment could also significantly enhance TAC3-induced SLα mRNA expression and the potentiating effect was dependent on TACR3 expression and activation of adenylate cyclase (AC)/cAMP/protein kinase A (PKA), phospholipase C (PLC)/inositol 1,4,5-triphosphate (IP3)/protein kinase C (PKC), and Ca2+/calmodulin (CaM)/calmodulin-dependent protein kinase II (CaMK-II) cascades. Besides, IGF-I-induced Akt phosphorylation but not MEK, extracellular signal-regulated kinase (ERK1/2), and P38MAPK phosphorylation was notably enhanced by TACR3 activation. These results, as a whole, suggest that the potentiating effect of IGFs on TAC3 gene products-induced SLα mRNA expression was mediated by TACR3 upregulation and functional crosstalk of post-receptor signaling in the pituitary.

Igf3 activates ß-catenin signaling to stimulate spermatogonial differentiation in zebrafish.

Follicle-stimulating hormone (Fsh) is a major regulator of spermatogenesis, targeting somatic cell functions in the testes. We reported previously that zebrafish Fsh promoted the differentiation of type A undifferentiated spermatogonia (Aund) by stimulating the production of factors that advance germ cell differentiation, such as androgens, insulin-like peptide 3 (Insl3) and insulin-like growth factor 3 (Igf3). In addition, Fsh also modulated the transcript levels of several other genes, including some belonging to the Wnt signaling pathway. Here, we evaluated if and how Fsh utilizes part of the canonical Wnt pathway to regulate the development of spermatogonia. We quantified the proliferation activity and relative section areas occupied by Aund and type A differentiating (Adiff) spermatogonia and we analyzed the expression of selected genes in response to recombinant proteins and pharmacological inhibitors. We found that from the three downstream mediators of Fsh activity we examined, Igf3, but not 11-ketotestosterone or Insl3, modulated the transcript levels of two ß-catenin sensitive genes (cyclinD1 and axin2). Using a zebrafish ß-catenin signaling reporter line, we showed that Igf3 activated ß-catenin signaling in type A spermatogonia and that this activation did not depend on the release of Wnt ligands. Pharmacological inhibition of the ß-catenin or of the phosphoinositide 3-kinase (PI3K) pathways revealed that Igf3 activated ß-catenin signaling in a manner involving PI3K to promote the differentiation of Aund to Adiff spermatogonia. This mechanism represents an intriguing example for a pituitary hormone like Fsh using Igf signaling to recruit the evolutionary conserved, local ß-catenin signaling pathway to regulate spermatogenesis.

Insulin-like growth factor (IGF)-like peptide and 20-hydroxyecdysone regulate the growth and development of the male genital disk through different mechanisms in the silkmoth, Bombyx mori.

It is well established that ecdysteroids play pivotal roles in the regulation of insect molting and metamorphosis. However, the mechanisms by which ecdysteroids regulate the growth and development of adult organs after pupation are poorly understood. Recently, we have identified insulin-like growth factor (IGF)-like peptides (IGFLPs), which are secreted after pupation under the control of 20-hydroxyecdysone (20E). In the silkmoth, Bombyx mori, massive amounts of Bombyx-IGFLP (BIGFLP) are present in the hemolymph during pupal-adult development, suggesting its importance in the regulation of adult tissue growth. Thus, we hypothesized that the growth and development of adult tissues including imaginal disks are regulated by the combined effects of BIGFLP and 20E. In this study, we investigated the growth-promoting effects of BIGFLP and 20E using the male genital disks of B. mori cultured ex vivo, and further analyzed the cell signaling pathways mediating hormone actions. We demonstrate that 20E induces the elongation of genital disks, that both hormones stimulate protein synthesis in an additive manner, and that BIGFLP and 20E exert their effects through the insulin/IGF signaling pathway and mitogen-activated protein kinase pathway, respectively. These results show that the growth and development of the genital disk are coordinately regulated by both BIGFLP and 20E.

Genome-wide interactions between FSH and insulin-like growth factors in the regulation of human granulosa cell differentiation.

Study question: Is the genome-wide response of human cumulus cells to FSH and insulin-like growth factors (IGFs) comparable to the response observed in undifferentiated granulosa cells (GCs)? Summary answer: FSH actions in human cumulus cells mimic those observed in preantral undifferentiated GCs from laboratory animals, and approximately half of the regulated genes are dependent on the simultaneous activation of the IGF1 receptor (IGF1R). What is known already: Animal studies have shown that FSH and the IGFs system are required for follicle growth and maturation. In humans, IGF levels in the follicular fluid correlate with patients' responses to IVF protocols. The main targets of FSH and IGFs in the ovary are the GCs; however, the genomic mechanisms involved in the response of GCs to these hormones are unknown. Study design, size, duration: Human cumulus cells isolated from IVF patients were cultured for 48 h in serum-free media in the presence of vehicle, FSH, IGF1R inhibitor or their combination. Participants/materials, setting, methods: Discarded cumulus cells were donated to research by reproductive-aged women undergoing IVF due to non-ovarian etiologies of infertility at a university-affiliated clinic. The effect of FSH and/or IGF1R inhibition on cumulus cell function was evaluated using Affymetrix microarrays, quantitative PCR, western blot, promoter assays and hormone level measurements. Main results and the role of chance: The findings demonstrate that human cumulus cells from IVF patients respond to FSH with the expression of genes known to be markers of the preantral to preovulatory differentiation of GCs. These results also demonstrate that ~50% of FSH-regulated genes require IGF1R activity and suggest that several aspects of follicle growth are coordinately regulated by FSH and IGFs in humans. This novel approach will allow for future mechanistic and molecular studies on the regulation of human follicle maturation. Large scale data: Data set can be accessed at Gene Expression Omnibus number GSE86427. Limitations, reasons for caution: Experiments were performed using primary human cumulus cells. This may not represent the response of intact follicles. Wider implications of the findings: Understanding the mechanisms involved in the regulation of GC differentiation by FSH and IGF in humans will contribute to improving treatments for infertility. Study funding/competing interest(s): The project was financed by the National Instituted of Health grant number R56HD086054 and R01HD057110 (C.S.). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. We have no competing interests to declare.

Effects and Mechanisms of Metformin on the Proliferation of Esophageal Cancer Cells In Vitro and In Vivo.

PURPOSE: The purpose of this study was to observe the effects of metformin on human esophageal cancer cell and to investigate its possible mechanisms. MATERIALS AND METHODS: Cell viability was detected by using a Cell Counting Kit-8, while cell cycle and apoptosis were assessed by flow cytometry and western blot was used to measure the expression of the related proteins. RNAi was used to knockout pyruvate kinase muscle isozyme 2 (PKM2). An Eca109 tumor model was established to evaluate the antitumor effect in vivo. Immunohistochemistry was determined based on the expression of PKM2 and Bim in tumor tissues. Tunnel was used to assess tumor cell apoptosis. RESULTS: Esophageal cancer cells viability was reduced after metformin treatment. The cell cycle was arrested in the G0/G1 phase, apoptosis was induced, caspase 3 was activated, caspase 9 was downregulated, and the pro-apoptotic protein Bim increased. Further study revealed that metformin could suppress the expression of insulin-like growth factor 1 receptor and its downstream proteins, phosphoinositide 3-kinase (PI3K), protein kinase B (AKT/PKB), phosphorylation of AKT (pAKT), mammalian target of rapamycin (mTOR), p70S6K, and PKM2. Insulin-like growth factor 1 partly reversed metfromin-induced apoptosis and attenuated the repression effect of metfomin to PI3K, pAKT, and PKM2. Knockout PKM2 resulted in the activation of caspase 3, down-regulation of caspase 9, and increased expression of Bim. In the Eca109 xenograft model, metformin significantly reduced tumor growth. Furthermore, we found that metformin treatment increased the rate of apoptosis, down-regulation of PKM2, and up-regulation of Bim in tumor tissues. CONCLUSION: Metformin restrained esophageal cancer cell proliferation partly by suppressing the PI3K/AKT/mTOR pathway.

PfIRR Interacts with HrIGF-I and Activates the MAP-kinase and PI3-kinase Signaling Pathways to Regulate Glycogen Metabolism in Pinctada fucata.

The insulin-induced mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways are major intracellular signaling modules and conserved among eukaryotes that are known to regulate diverse cellular processes. However, they have not been investigated in the mollusk species Pinctada fucata. Here, we demonstrate that insulin-related peptide receptor of P. fucata (pfIRR) interacts with human recombinant insulin-like growth factor I (hrIGF-I), and stimulates the MAPK and PI3K signaling pathways in P. fucata oocytes. We also show that inhibition of pfIRR by the inhibitor PQ401 significantly attenuates the basal and hrIGF-I-induced phosphorylation of MAPK and PI3K/Akt at amino acid residues threonine 308 and serine 473. Furthermore, our experiments show that there is cross-talk between the MAPK and PI3K/Akt pathways, in which MAPK kinase positively regulates the PI3K pathway, and PI3K positively regulates the MAPK cascade. Intramuscular injection of hrIGF-I stimulates the PI3K and MAPK pathways to increase the expression of pfirr, protein phosphatase 1, glucokinase, and the phosphorylation of glycogen synthase, decreases the mRNA expression of glycogen synthase kinase-3 beta, decreases glucose levels in hemocytes, and increases glycogen levels in digestive glands. These results suggest that the MAPK and PI3K pathways in P. fucata transmit the hrIGF-I signal to regulate glycogen metabolism.

Releasing prophase arrest in zebrafish oocyte: synergism between maturational steroid and Igf1.

Binding of 17ß-estradiol (E2) to novel G-protein coupled receptor, Gper1, promotes intra-oocyte adenylyl cyclase activity and transactivates epidermal growth factor receptor to ensure prophase-I arrest. Although involvement of either membrane progestin receptor (mPR) or Igf system has been implicated in regulation of meiosis resumption, possibility of concurrent activation and potential synergism between 17α,20ß-dihydroxy-4-pregnen-3-one (DHP)- and Igf-mediated signalling cascades in alleviating E2 inhibition of oocyte maturation (OM) has not been investigated. Here using zebrafish (Danio rerio) defolliculated oocytes, we examined the effect of DHP and Igf1, either alone or in combination, in presence or absence of E2, on OM in vitro. While priming of denuded oocytes with E2 blocked spontaneous maturation, co-treatment with DHP (3 nM) and Igf1 (10 nM), but not alone, reversed E2 inhibition and promoted a robust increase in germinal vesicle breakdown (GVBD). Although stimulation with either Igf1 or DHP promoted Akt phosphorylation, pharmacological inhibition of PI3K/Akt signalling prevented Igf1-induced GVBD but delayed DHP action till 4-5 h of incubation. Moreover, high intra-oocyte cAMP attenuates both DHP and Igf1-mediated OM and co-stimulation with DHP and Igf1 could effectively reverse E2 action on PKA phosphorylation. Interestingly, data from in vivo studies reveal that heightened expression of igf1, igf3 transcripts in intact follicles corresponded well with elevated phosphorylation of Igf1r and Akt, mPRa immunoreactivity, PKA inhibition and accelerated GVBD response just prior to ovulation. This indicates potential synergism between maturational steroid and Igf1 which might have physiological relevance in overcoming E2 inhibition of meiosis resumption in zebrafish oocytes.

Mesenchymal Stem Cells Exhibit Regulated Exocytosis in Response to Chemerin and IGF.

Mesenchymal stem cells (MSCs) play important roles in tissue repair and cancer progression. Our recent work suggests that some mesenchymal cells, notably myofibroblasts exhibit regulated exocytosis resembling that seen in neuroendocrine cells. We now report that MSCs also exhibit regulated exocytosis. Both a G-protein coupled receptor agonist, chemerin, and a receptor tyrosine kinase stimulant, IGF-II, evoked rapid increases in secretion of a marker protein, TGFßig-h3. The calcium ionophore, ionomycin, also rapidly increased secretion of TGFßig-h3 while inhibitors of translation (cycloheximide) or secretory protein transport (brefeldin A) had no effect, indicating secretion from preformed secretory vesicles. Inhibitors of the chemerin and IGF receptors specifically reduced the secretory response. Confocal microscopy of MSCs loaded with Fluo-4 revealed chemerin and IGF-II triggered intracellular Ca2+ oscillations requiring extracellular calcium. Immunocytochemistry showed co-localisation of TGFßig-h3 and MMP-2 to secretory vesicles, and transmission electron-microscopy showed dense-core secretory vesicles in proximity to the Golgi apparatus. Proteomic studies on the MSC secretome identified 64 proteins including TGFßig-h3 and MMP-2 that exhibited increased secretion in response to IGF-II treatment for 30min and western blot of selected proteins confirmed these data. Gene ontology analysis of proteins exhibiting regulated secretion indicated functions primarily associated with cell adhesion and in bioassays chemerin increased adhesion of MSCs and adhesion, proliferation and migration of myofibroblasts. Thus, MSCs exhibit regulated exocytosis that is compatible with an early role in tissue remodelling.

Fsh Stimulates Spermatogonial Proliferation and Differentiation in Zebrafish via Igf3.

Growth factors modulate germ line stem cell self-renewal and differentiation behavior. We investigate the effects of Igf3, a fish-specific member of the igf family. Fsh increased in a steroid-independent manner the number and mitotic index of single type A undifferentiated spermatogonia and of clones of type A differentiating spermatogonia in adult zebrafish testis. All 4 igf gene family members in zebrafish are expressed in the testis but in tissue culture only igf3 transcript levels increased in response to recombinant zebrafish Fsh. This occurred in a cAMP/protein kinase A-dependent manner, in line with the results of studies on the igf3 gene promoter. Igf3 protein was detected in Sertoli cells. Recombinant zebrafish Igf3 increased the mitotic index of type A undifferentiated and type A differentiating spermatogonia and up-regulated the expression of genes related to spermatogonial differentiation and entry into meiosis, but Igf3 did not modulate testicular androgen release. An Igf receptor inhibitor blocked these effects of Igf3. Importantly, the Igf receptor inhibitor also blocked Fsh-induced spermatogonial proliferation. We conclude that Fsh stimulated Sertoli cell production of Igf3, which promoted via Igf receptor signaling spermatogonial proliferation and differentiation and their entry into meiosis. Because previous work showed that Fsh also released spermatogonia from an inhibitory signal by down-regulating anti-Müllerian hormone and by stimulating androgen production, we can now present a model, in which Fsh orchestrates the activity of stimulatory (Igf3, androgens) and inhibitory (anti-Müllerian hormone) signals to promote spermatogenesis.

Stromal transcriptional profiles reveal hierarchies of anatomical site, serum response and disease and identify disease specific pathways.

Synovial fibroblasts in persistent inflammatory arthritis have been suggested to have parallels with cancer growth and wound healing, both of which involve a stereotypical serum response programme. We tested the hypothesis that a serum response programme can be used to classify diseased tissues, and investigated the serum response programme in fibroblasts from multiple anatomical sites and two diseases. To test our hypothesis we utilized a bioinformatics approach to explore a publicly available microarray dataset including rheumatoid arthritis (RA), osteoarthritis (OA) and normal synovial tissue, then extended those findings in a new microarray dataset representing matched synovial, bone marrow and skin fibroblasts cultured from RA and OA patients undergoing arthroplasty. The classical fibroblast serum response programme discretely classified RA, OA and normal synovial tissues. Analysis of low and high serum treated fibroblast microarray data revealed a hierarchy of control, with anatomical site the most powerful classifier followed by response to serum and then disease. In contrast to skin and bone marrow fibroblasts, exposure of synovial fibroblasts to serum led to convergence of RA and OA expression profiles. Pathway analysis revealed three inter-linked gene networks characterising OA synovial fibroblasts: Cell remodelling through insulin-like growth factors, differentiation and angiogenesis through _3 integrin, and regulation of apoptosis through CD44. We have demonstrated that Fibroblast serum response signatures define disease at the tissue level, and that an OA specific, serum dependent repression of genes involved in cell adhesion, extracellular matrix remodelling and apoptosis is a critical discriminator between cultured OA and RA synovial fibroblasts.

Recombinant growth factor mixtures induce cell cycle progression and the upregulation of type I collagen in human skin fibroblasts, resulting in the acceleration of wound healing processes.

Application of growth factor mixtures has been used for wound healing and anti-wrinkles agents. The aim of this study was to evaluate the effect of recombinant growth factor mixtures (RGFM) on the expression of cell cycle regulatory proteins, type I collagen, and wound healing processes of acute animal wound models. The results showed that RGFM induced increased rates of cell proliferation and cell migration of human skin fibroblasts (HSF). In addition, expression of cyclin D1, cyclin E, cyclin-dependent kinase (Cdk)4, and Cdk2 proteins was markedly increased with a growth factor mixtures treatment in fibroblasts. Expression of type I collagen was also increased in growth factor mixtures-treated HSF. Moreover, growth factor mixtures-induced the upregulation of type I collagen was associated with the activation of Smad2/3. In the animal model, RGFM-treated mice showed accelerated wound closure, with the closure rate increasing as early as on day 7, as well as re-epithelization and reduced inflammatory cell infiltration than phosphate-buffered saline (PBS)-treated mice. In conclusion, the results indicated that RGFM has the potential to accelerate wound healing through the upregulation of type I collagen, which is partly mediated by activation of Smad2/3-dependent signaling pathway as well as cell cycle progression in HSF. The topical application of growth factor mixtures to acute and chronic skin wound may accelerate the epithelization process through these molecular mechanisms.

An insulin-like growth factor homologue of Singapore grouper iridovirus modulates cell proliferation, apoptosis and enhances viral replication.

Insulin-like growth factors (IGFs) play crucial roles in regulating cell differentiation, proliferation and apoptosis. In this study, a novel IGF homologue gene (IGF-like) encoded by Singapore grouper iridovirus (SGIV) ORF062R (termed SGIV-IGF), was cloned and characterized. The coding region of SGIV-IGF is 771 bp in length, with a variable number of tandem repeats (VNTR) locus at the 3'-end. We cloned one isoform of this novel gene, 582 bp in length, containing the predicted IGF domain and 3.6 copy numbers of the 27 bp repeat unit. SGIV-IGF was an early transcribed gene during viral infection, and SGIV-IGF was distributed predominantly in the cytoplasm with a diffused granular appearance. Intriguingly, overexpression of SGIV-IGF was able to promote the growth of grouper embryonic cells (GP cells) by promoting G1/S phase transition, which was at least partially dependent on its 3'-end VNTR locus. Furthermore, viral titre assay and real-time quantitative PCR (RT-qPCR) analysis proved that SGIV-IGF could promote SGIV replication in grouper cells. In addition, overexpression of SGIV-IGF mildly facilitated apoptosis in SGIV-infected non-host fathead minnow (FHM) cells. Together, our study demonstrated a novel functional gene of SGIV which may regulate viral replication and cellular processes through multiple mechanisms that appear to be cell type-dependent.

Proliferative status regulates HDAC11 mRNA abundance in nontransformed fibroblasts.

Histone deacetylases (HDACs) are important determinants of gene transcription and other biological processes. HDAC11 is one of the least characterized HDACs and is the only member of the class IV HDAC family. Our studies examined the events that control the expression of the HDAC11 transcript. We show that platelet-derived growth factor (PDGF) rapidly reduces the abundance of HDAC11 mRNA when added to density-arrested Balb/c-3T3 cells, which are nontransformed fibroblasts. Reduction required mRNA and protein synthesis, but not AKT or ERK activity, and resulted from accelerated turnover of the HDAC11 transcript. Reduction was transient in cells receiving PDGF alone but sustained in cells receiving both PDGF and platelet-poor plasma, which together promote G0/G1 traverse and S phase entry. Plasma alone did not appreciably reduce HDAC11 mRNA abundance, nor did epidermal growth factor, insulin-like growth factor, or insulin. HDAC11 mRNA accumulated in Balb/c-3T3 cells exiting the cell cycle due to density-dependent growth inhibition or serum deprivation. Of note, HDAC11 mRNA did not accumulate in a spontaneously transformed Balb/c-3T3 clonal variant (clone 2) that does not density arrest. The HDAC11 promoter was active in Balb/c-3T3 but not clone 2 cells; inactivity in clone 2 cells did not result from methylation of CpG islands. Overexpression of HDAC11 inhibited the cell cycle progression of both transformed and nontransformed fibroblasts. Our studies identify the HDAC11 transcript as a PDGF target and show that HDAC11 mRNA abundance correlates inversely with proliferative status.

William H. Daughaday and the foundations of modern research into growth hormone and the insulin-like growth factors.

This vignette summarizes some of the scientific accomplishments of Dr. William H. Daughaday, a founder of modern research into the biological effects of growth hormone and the insulin-like growth factors, and formulator of the somatomedin hypothesis of GH actions on growth.