Areg mRNA Research Articles

Amphiregulin Exerts Proangiogenic Effects in Developing Murine Lungs.

Interrupted lung angiogenesis is a hallmark of bronchopulmonary dysplasia (BPD); however, druggable targets that can rescue this phenotype remain elusive. Thus, our investigation focused on amphiregulin (Areg), a growth factor that mediates cellular proliferation, differentiation, migration, survival, and repair. While Areg promotes lung branching morphogenesis, its effect on endothelial cell (EC) homeostasis in developing lungs is understudied. Therefore, we hypothesized that Areg promotes the proangiogenic ability of the ECs in developing murine lungs exposed to hyperoxia. Lung tissues were harvested from neonatal mice exposed to normoxia or hyperoxia to determine Areg expression. Next, we performed genetic loss-of-function and pharmacological gain-of-function studies in normoxia- and hyperoxia-exposed fetal murine lung ECs. Hyperoxia increased Areg mRNA levels and Areg+ cells in whole lungs. While Areg expression was increased in lung ECs exposed to hyperoxia, the expression of its signaling receptor, epidermal growth factor receptor, was decreased, indicating that hyperoxia reduces Areg signaling in lung ECs. Areg deficiency potentiated hyperoxia-mediated anti-angiogenic effects. In contrast, Areg treatment increased extracellular signal-regulated kinase activation and exerted proangiogenic effects. In conclusion, Areg promotes EC tubule formation in developing murine lungs exposed to hyperoxia.

MiR-33a-3p regulates METTL3-mediated AREG stability and alters EMT to inhibit pancreatic cancer invasion and metastasis

Recent studies have shown that amphoteric regulatory protein (AREG), a member of the epidermal growth factor (EGF) family, is expressed in many cancers and is an independent prognostic indicator for patients with pancreatic cancer, but whether AREG is regulated at the epigenetic level to promote the development of pancreatic cancer (PC) has not been elucidated. Our results support the notion that AREG is overexpressed in pancreatic cancer tissues and cell lines. Functionally, the deletion of AREG impedes pancreatic cancer (PC) cell proliferation, migration, and invasion. In addition, we identified and validated that methyltransferase-like 3 (METTL3) induced the m6A modification on AREG and facilitated the stability of AREG mRNA after sequencing. Additionally, we obtained experimental evidence that miR-33a-3p targets and inhibits METTL3 from taking action, as predicted by using the miRDB and RNAinter. Remediation experiments showed that miR-33a-3p inhibits PC progression through METTL3. In summary, this research reveals that miR-33a-3p inhibits m6A-induced stabilization of AREG by targeting METTL3, which plays a key role in the aggressive progression of PC. AREG could be a potential target for PC treatment.

Oocyte secreted factors control genes regulating FSH signaling and the maturation cascade in cumulus cells: the oocyte is not in a hurry.

To assess the effects of the oocyte on mRNA abundance of FSHR, AMH and major genes of the maturation cascade (AREG, EREG, ADAM17, EGFR, PTGS2, TNFAIP6, PTX3, and HAS2) in bovine cumulus cells. (1) Intact cumulus-oocyte complexes, (2) microsurgically oocytectomized cumulus-oolema complexes (OOX), and (3) OOX + denuded oocytes (OOX+DO) were subjected to in vitro maturation (IVM) stimulated with FSH for 22 h or with AREG for 4 and 22 h. After IVM, cumulus cells were separated and relative mRNA abundance was measured by RT-qPCR. After 22 h of FSH-stimulated IVM, oocytectomy increased FSHR mRNA levels (p=0.005) while decreasing those of AMH (p=0.0004). In parallel, oocytectomy increased mRNA abundance of AREG, EREG, ADAM17, PTGS2, TNFAIP6, and PTX3, while decreasing that of HAS2 (p<0.02). All these effects were abrogated in OOX+DO. Oocytectomy also reduced EGFR mRNA levels (p=0.009), which was not reverted in OOX+DO. The stimulatory effect of oocytectomy on AREG mRNA abundance (p=0.01) and its neutralization in OOX+DO was again observed after 4 h of AREG-stimulated IVM. After 22 h of AREG-stimulated IVM, oocytectomy and addition of DOs to OOX caused the same effects on gene expression observed after 22 h of FSH-stimulated IVM, except for ADAM17 (p<0.025). These findings suggest that oocyte-secreted factors inhibit FSH signaling and the expression of major genes of the maturation cascade in cumulus cells. These may be important actions of the oocyte favoring its communication with cumulus cells and preventing premature activation of the maturation cascade.

Donor-Derived Amphiregulin Drives CD4+ T Cell Expansion and Promotes Tissue Pathology after Experimental Allogeneic BMT

Amphiregulin (Areg), a ligand for the epidermal growth factor receptor (EGFR), contributes to cell proliferation and survival. Work in recent years has demonstrated that immune cells such as Tregs and ILC2s can produce Areg, leading to tissue repair after injury. In the bone marrow transplant (BMT) setting, clinical studies have reported that high concentrations of plasma Areg may predict for poor prognosis acute GVHD. However, impacts of Areg/EGFR signaling within the immune system are poorly understood. We thus investigated the role of T cell-derived Areg following allogeneic (allo)-BMT. To evaluate Areg expression in donor T cells, we performed C57BL/6 into B6D2F1 (B6 BDF1) MHC-mismatched allo-BMT and found that donor CD4+ T cells produced Areg post-transplant. We initially hypothesized that T cell-derived Areg may contribute to tissue repair after BMT. To investigate the effects of T cell-derived Areg in the transplant setting, we performed B6 BDF1 allo-BMT using T cells from Aregfl/fl x CD4-Cre (Areg-/-) or Aregfl/fl x Foxp3-Cre (AregΔFoxp3) mice, lacking Areg in all T cells (Areg-/-) or specifically in regulatory T cells (AregΔFoxp3). Unexpectedly, recipients of Areg-/- T cells demonstrated reduced mortality compared to recipients of wild-type (WT) T cells (Figure, left). This observation was recapitulated in a distinct B6 BALB/c allo-BMT model (not shown). Examining the effects of Areg deficiency at the tissue level, recipients of Areg-/- T cells demonstrated reduced GVHD pathology in liver, ileum, and colon ten days post-BMT, while recipients of AregΔFoxp3 T cells showed no difference compared to controls. Consistent with the GVHD histopathology, intestinal stem cell frequencies were substantially preserved in the small intestines from recipients transplanted with Areg-/- T cells compared to recipients receiving WT T cells or AregΔFoxp3 T cells. These data suggested that conventional T cell-derived Areg may contribute to tissue damage in GVHD. To assess the mechanisms by which T cell-derived Areg contributes to GVHD severity, we analyzed donor T cells in spleen, mesenteric lymph nodes, and lamina propria post-BMT. Areg-/- donor CD4+ T cells demonstrated impaired expansion while donor CD8+ T cell expansion remained intact. Areg-/- donor CD4+ T cells also demonstrated a reduced Th1 response as evidenced by decreased IFNg expression. In addition, co-culture in vitro and co-transfer in vivo of WT and Areg-/- CD4+ T cells indicated that the reduced expansion of Areg-/- donor CD4+ T cells was cell-intrinsic and likely occurring in an autocrine Areg-dependent fashion. Notably, these observations were recapitulated following allo-BMT using EGFRfl/fl x CD4-Cre (EGFR-/-) donor T cells: recipients transplanted with EGFR-/- donor T cells demonstrated reduced donor CD4+ T cell expansion and improved survival post-transplant (Figure, right). To further investigate the mechanisms driving Areg-dependent CD4+ T cell expansion, we compared WT and Areg-/-B6 T cells following ex vivo activation with anti-CD3 and anti-CD28 antibodies or activation with allo BALB/c-derived dendritic cells. Compared to WT CD4+ T cells, Areg-deficient CD4+ T cells exhibited reduced proliferation, which was restored by supplementation with recombinant Areg. Additionally, we found that naive CD4+ T cells increased Egfr and Areg mRNA expression upon T cell receptor stimulation. Moreover, Areg inhibition using anti-Areg neutralizing antibodies impaired proliferation of WT naive CD4+ T cells after activation, reducing their expansion upon activation ex vivo. To better understand how Areg was impacting activation-induced CD4+ T cell expansion, we examined downstream targets of EGFR signaling and observed that Areg inhibition resulted in impairment of Akt phosphorylation and reduced glucose uptake in naive CD4+ T cells following activation. Finally, despite the reduction in proliferation and GVHD pathogenicity, Areg-/- donor T cells demonstrated intact GVL activity against A20 lymphoma cells, resulting in an improved overall survival following BMT with tumor challenge. In summary, we found that CD4+ T cell-derived Areg contributes to CD4+ T cell proliferation after activation, leading to increased T cell expansion, tissue damage, and GVHD-related mortality after allo-BMT. Careful inhibition of the T cell Areg/EGFR axis may represent a novel therapeutic target for prevention or amelioration of GVHD. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Inflammatory and Proliferative Pathway Activation in Human Esophageal Myofibroblasts Treated with Acidic Bile Salts.

Subepithelial human esophageal myofibroblasts (HEMFs) in gastroesophageal reflux disease (GERD) are exposed to luminal contents via impaired squamous epithelium barrier integrity. The supernatant of HEMFs treated with acidic bile salts reflective of in vivo reflux increases squamous epithelial thickness. We aimed to identify the involved mechanisms using an unbiased approach. Acidic-bile-salt-treated primary HEMF cultures (n = 4) were submitted for RNA-Seq and analyzed with Partek Flow followed by Ingenuity Pathway Analysis (IPA). A total of 1165 molecules (579 downregulated, 586 upregulated) were differentially expressed, with most top regulated molecules either extracellular or in the plasma membrane. Increases in HEMF CXCL-8, IL-6, AREG, and EREG mRNA, and protein secretion were confirmed. Top identified canonical pathways were agranulocyte and granulocyte adhesion and diapedesis, PI3K/AKT signaling, CCR5 signaling in macrophages, and the STAT3 pathway. Top diseases and biological functions were cellular growth and development, hematopoiesis, immune cell trafficking, and cell-mediated response. The targets of the top upstream regulator ErbB2 included CXCL-8, IL-6, and AREG and the inhibition of CXCL-8 in the HEMF supernatant decreased squamous epithelial proliferation. Our work shows an inflammatory/immune cell and proliferative pathways activation in HEMFs in the GERD environment and identifies CXCL-8 as a HEMF-derived chemokine with paracrine proliferative effects on squamous epithelium.

Celastrol attenuates amphiregulin expression by inhibiting MAPK signaling pathway in triple-negative breast cancer cells

BackgroundAmphiregulin (AREG) plays an essential role in the metastasis and angiogenesis of various cancers. PurposeThe objective of this study was to investigate the pharmacological effects of celastrol on AREG expression in triple-negative breast cancer (TNBC) cells. MethodsWe analyzed the prognostic value of AREG, EGF, TGFA, and HB-EGF mRNA expression in TNBC patients. Cell proliferation was analyzed by MTT assay and colony-forming assay. Levels of protein and gene expression were analyzed by ELISA, Western blotting, confocal microscopy, and real-time PCR, respectively. The expression of angiogenic proteins was analyzed by using the Human angiogenesis array kit. Cell invasiveness was analyzed by Boyden chamber assay. ResultsOur results showed that abnormal AREG induction remarkably decreased the relapse-free survival (RFS) rate of TNBC patients, whereas epidermal growth factor (EGF), transforming growth factor-alpha (TGFA), or heparin-binding EGF-like growth factor (HBEGF) demonstrated no effect on RFS rate. In addition, the growth of TNBC cells was prevented by drugs targeting EGFR such as neratinib or lapatinib. On the contrary, cell growth and invasion were enhanced by AREG treatment. Interestingly, celastrol reduced AREG expression and the growth of TNBC cells. Furthermore, we found that levels of AREG expression were reduced by a potent inhibitor of MEK1/2, binimetinib (BIN). Our results showed that celastrol significantly decreased the levels of ERK phosphorylation in TNBC cells. Finally, we observed that celastrol also suppressed the AREG-induced EGFR signaling pathway. Taken together, these results suggest that celastrol can downregulate AREG expression by inhibiting the MEK/ERK signaling pathway. In addition, the AREG-induced EGFR signaling pathway was inhibited by celastrol. ConclusionCelastrol can be an effective drug for treating TNBC by inhibiting AREG/EGFR signaling pathways.

SAMiRNA Targeting Amphiregulin Alleviate Total-Body-Irradiation-Induced Renal Fibrosis.

Fibrosis is a serious unintended side effect of radiation therapy. In this study, we aimed to investigate whether amphiregulin (AREG) plays a critical role in fibrosis development after total-body irradiation (TBI). We found that the expression of AREG and fibrotic markers, such as α-smooth muscle actin (α-SMA) and collagen type I alpha 1 (COL1α1), was elevated in the kidneys of 6 Gy TBI mice. Expression of AREG and α-SMA was mainly elevated in the proximal and distal tubules of the kidney in response to TBI, which was confirmed by immunofluorescence staining. Knockdown of Areg mRNA using self-assembled-micelle inhibitory RNA (SAMiRNA) significantly reduced the expression of fibrotic markers, including α-SMA and COL1α1, and inflammatory regulators. Finally, intravenous injections of SAMiRNA targeting mouse Areg mRNA (SAMiRNA-mAREG) diminished radiation-induced collagen accumulation in the renal cortex and medulla. Taken together, the results of the present study suggest that blocking of AREG signaling via SAMiRNA-mAREG treatment could be a promising therapeutic approach to alleviate radiation-induced kidney fibrosis.

Improvement in early antral follicle development and gene expression modulation prior to follicle aspiration in bovine cumulus-oocyte complexes by equine chorionic gonadotropin

We aimed to evaluate the morphological ovarian response to equine chorionic gonadotropin (eCG) prior to ovum pick-up (OPU) and its effects on the molecular phenotype of immature cumulus-oocyte complexes (COCs) from Nelore cow (Bos indicus) donors. To this end, 20 Nelore cows were distributed randomly into the synchronized-OPU (Sync-OPU) and synchronized plus stimulated-OPU (Sync + eCG-OPU) groups using a cross-over experimental design, as each cow was used in both treatments. On a random day of the estrus cycle (Day 0), all cows received an intravaginal implant with 1.0 g of progesterone and 2 mg IM of estradiol benzoate. On the morning of Day 3, only the Sync + eCG-OPU group received 400 IU of eCG IM. On the morning of Day 5, the P4 device was removed and OPU was conducted in both groups. Before OPU management, ultrasonography was used to identify and measure the follicles. The aspirated COCs were morphologically classified based on their cumulus cells (CC) layers and the texture of the ooplasm. The COCs classified as Grade 1, Grade 2, and Grade 3 were considered viable and used for the assessment of quality markers. Oocytes and CC were mechanically separated from pools of 25 immature COCs of the Sync-OPU and Sync + eCG-OPU groups immediately after the follicular aspiration and stored at −80 °C until RNA extraction. Relative quantification of several markers for oocyte quality was assessed by RT-qPCR. The eCG treatment increased the number of follicles sized 3.0–5.0 mm and >5.0 mm compared to that in Sync-OPU group. Moreover, the protocol with eCG improved the total number of oocytes and the number of viable oocytes, which is related to a high number of oocytes in Grade 3. Regarding the impact on transcriptional regulation in immature oocytes, the mRNA encoding BMP15, SMAD1, SMAD2, SMAD3, ACACA, and CPT1A was upregulated in Sync + eCG-OPU compared with the Sync-OPU group. Moreover, the relative mRNA abundance of CTSZ, a member of the cathepsins family functionally related to reduced oocyte competence, was lower in the Sync + eCG-OPU group than in the Sync-OPU group. In addition, CC CTSB, CTSS, and CTSK mRNA abundances were lower in the Sync + eCG-OPU group than in the Sync-OPU group. However, the relative abundance of AREG and EREG mRNA was higher in CC recovered from cows stimulated with eCG. In conclusion, the eCG approach addressing follicular stimulation in Nelore cows had a positive impact on early antral follicle development, followed by a positive morphological and molecular phenotype in bovine COCs.

Inhibitory Effects and Potential Mechanism of Yokukansankachimpihange on SDS-induced Itch-associated Responses in Mice

Yokukansankachimpihange (YKSCH) is a Chinese formula proven in Japan to be an effective treatment for pruritus in patients with atopic dermatitis or chronic urticaria and dry eczema with psychoneurotic symptoms, such as insomnia. Sodium dodecyl sulfate (SDS) may cause skin irritation in both humans and animals. Adverse effects, such as skin dryness, barrier destruction, dermatitis, and pruritus, developed following the repeated application of 10% SDS to a local site. Therefore, the present study investigated the antipruritic effects of YKSCH on skin irritation induced by a repeated SDS stimulation in mice and attempted to elucidate the underlying mechanism of action. ICR mice were stimulated with 10% SDS and co-treated with oral YKSCH extract for four days. Only oral YKSCH extract was administered on the fifth day. Scratching behavior was observed 24 h after the last application of SDS. Intraepidermal nerve growth was investigated by an immunofluorescence analysis. NGF concentrations in the epidermis were measured using an enzyme-linked immunosorbent assay. The expression of amphiregulin (Areg) and semaphorin 3A (Sema3A) was assessed by quantitative real-time PCR. The oral administration of YKSCH (200-400 mg/kg) dose-dependently suppressed pruritus, significantly reduced intraepidermal nerve growth, and down-regulated epidermal Areg mRNA expression in SDS-treated mice. These results suggest that YKSCH exerts antipruritic effects against SDS-induced pruritus in mice. The mechanism of action of YKSCH may involve reductions in intraepidermal nerve density due to the down-regulated expression of Areg.

Exposure to a Glyphosate-based Herbicide Alters the Expression of Key Regulators of Mammary Gland Development on Pre-pubertal Male Rats

We previously reported that exposure during gestation and lactation to a low dose of glyphosate-based herbicide (GBH) reduced the area and perimeter of male offspring mammary gland at postnatal day 60 (PND60), whereas a higher dose increased the longitudinal growth of the gland. Here, our aim was to assess whether perinatal exposure to GBH exhibits endocrine disruptive action in male mammary gland at an early time point (pre-puberty), which could be related to the changes observed after puberty. We also wanted to explore whether an early evaluation of the male rat mammary gland is appropriate to assess exposure to potential endocrine disrupting chemicals (EDCs). Pregnant rats were orally exposed, through the diet, to vehicle (saline solution), 3.5 or 350 mg/kg/day of GBH from gestational day 9 until weaning. At PND21, the male offspring were euthanized, and mammary gland samples were collected. The histology and proliferation index of the mammary glands were evaluated, and the mRNA expression of estrogen (ESR1) and androgen (AR) receptors, cyclin D1 (Ccnd1), amphiregulin (Areg), insulin-like growth factor 1 (IGF1), epidermal growth factor receptor (EGFR) and IGF1 receptor (IGF1R) were assessed. Moreover, the phosphorylated-Erk1/2 (p-ERK1/2) protein expression was determined. No differences were observed in mammary epithelial structures and AR expression between experimental groups; however, the proliferation index was reduced in GBH3.5-exposed males. This result was associated with decreased ESR1, Ccnd1, Areg, IGF1, EGFR and IGF1R mRNA expressions, as well as reduced p-Erk1/2 protein expression in these animals. ESR1, Ccnd1, IGF1R and EGFR expressions were also reduced in GBH350-exposed males. In conclusion, the mammary gland development of pre-pubertal male rats is affected by perinatal exposure to GBH. Although further studies are still needed to understand the molecular mechanisms involved in GBH350 exposure, the present results may explain the alterations observed in mammary gland growth of post-pubertal males exposed to low doses of GBH. Our results also suggest that early evaluation of the male rat mammary gland is useful in assessing exposure to potential EDCs. However, analysis of EDCs effects at later time points should not be excluded.

Cumulus cell pappalysin-1, luteinizing hormone/choriogonadotropin receptor, amphiregulin and hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 mRNA levels associate with oocyte developmental competence and embryo outcomes.

To determine whether a selected set of mRNA biomarkers expressed in individual cumulus granulosa cell (CC) masses show association with oocyte developmental competence, embryo ploidy status, and embryo outcomes. This prospective observational cohort pilot study assessed levels of mRNA biomarkers in 163 individual CC samples from 15 women stimulated in antagonist cycles. Nineteen mRNA biomarker levels were measured by real-time PCR and related to the development of their corresponding individually cultured oocytes and subsequent embryos, embryo ploidy status, and live birth outcomes. PAPPA mRNA levels were significantly higher in CC from oocytes that led to euploid embryos resulting in live births and aneuploid embryos compared to immature oocytes by ANOVA. LHCGR mRNA levels were significantly higher in CC of oocytes resulting in embryos associated with live birth compared to immature oocytes and oocytes resulting in arrested embryos by ANOVA. Using a general linearized mixed model to assess ploidy status, CC HSD3B mRNA levels in oocytes producing euploid embryos were significantly lower than other oocyte outcomes, collectively. When transferred euploid embryos outcomes were analyzed by ANOVA, AREG mRNA levels were significantly lower and PAPPA mRNA levels significantly higher in CC from oocytes that produced live births compared to transferred embryos that did not form a pregnancy. Collectively, PAPPA, LHCGR, and AREG mRNA levels in CC may be able to identify oocytes with the best odds of resulting in a live birth, and HSD3B1 mRNA levels may be able to identify oocytes capable of producing euploid embryos.

ADAM17 is essential for ectodomain shedding of the EGF-receptor ligand amphiregulin.

The epidermal growth factor (EGF)‐receptor ligand amphiregulin (AREG) is a potent growth factor implicated in proliferative skin diseases and in primary and metastatic epithelial cancers. AREG, synthesized as a propeptide, requires conversion to an active peptide by metalloproteases by a process known as ectodomain shedding. Although (ADAM17) a disintegrin and metalloprotease 17 is a key sheddase of AREG, ADAM8‐, ADAM15‐, and batimastat (broad metalloprotease inhibitor)‐sensitive metalloproteases have also been implicated in AREG shedding. In the present study, using a curly bare (Rhbdf2 cub) mouse model that shows loss‐of‐hair, enlarged sebaceous gland, and rapid cutaneous wound‐healing phenotypes mediated by enhanced Areg mRNA and protein levels, we sought to identify the principal ectodomain sheddase of AREG. To this end, we generated Rhbdf2 cub mice lacking ADAM17 specifically in the skin and examined the above phenotypes of Rhbdf2 cub mice. We find that ADAM17 deficiency in the skin of Rhbdf2 cub mice restores a full hair coat, prevents sebaceous gland enlargement, and impairs the rapid wound‐healing phenotype observed in Rhbdf2 cub mice. Furthermore, in vitro, stimulated shedding of AREG is abolished in Rhbdf2 cub mouse embryonic keratinocytes lacking ADAM17. Thus, our data support previous findings demonstrating that ADAM17 is the major ectodomain sheddase of AREG.

Transgenic mice specifically expressing amphiregulin in white adipose tissue showed less adipose tissue mass.

To determine adipocytokines that play a regulatory role during obesity development, we explored the genes that encode growth factors and investigated the physiological functions for adipose tissue development. Here, we isolated amphiregulin (Areg) gene whose expression was significantly up-regulated in obese adipose tissues. Areg mRNA level was positively correlated with macrophage marker gene expression in adipose tissues in vivo. Unexpectedly, Areg transgenic mice showed less adipose tissue mass with increased mRNA expression levels of Tnf-α and peroxisome proliferator-activated receptor γ coactivator 1α (Pgc-1α) and delayed white adipose tissue development during the convalescent stage in a dextran sodium sulfate-induced colitis model. This study showed that Areg mRNA expression was significantly up-regulated in obese adipose tissues and over-expression of Areg in white adipose tissue caused less adipose tissue mass.

Growth Arrest Specific-1 (GAS1) Is a C/EBP Target Gene That Functions in Ovulation and Corpus Luteum Formation in Mice.

Ovulation and luteinization are initiated in preovulatory follicles by the luteinizing hormone (LH) surge; however, the signaling events that mediate LH actions in these follicles remain incompletely defined. Two key transcription factors that are targets of LH surge are C/EBPalpha and C/EBPbeta, and their depletion in granulosa cells results in complete infertility. Microarray analyses of these mutant mice revealed altered expression of a number of genes, including growth arrest specific-1 (Gas1). To investigate functions of Gas1 in ovulation- and luteinization-related processes, we crossed Cyp19a1-Cre and Gas1flox/flox mice to conditionally delete Gas1 in granulosa and cumulus cells. While expression of Gas1 is dramatically increased in granulosa and cumulus cells around 12–16 h post-human chorionic gonadotropin (hCG) stimulation in wild-type mice, this increase is abolished in Cebpa/b double mutant and in Gas1 mutant mice. GAS1 is also dynamically expressed in stromal cells of the ovary independent of C/EBPalpha/beta. Female Gas1 mutant mice are fertile, exhibit enhanced rates of ovulation, increased fertility, and higher levels of Areg and Lhcgr mRNA in granulosa cells. The morphological appearance and vascularization of corpora lutea appeared normal in these mutant females. Interestingly, levels of mRNA for a number of genes (Cyp11a1, Star, Wnt4, Prlr, Cd52, and Sema3a) associated with luteinization are decreased in corpora lutea of Gas1 mutant mice as compared with controls at 24 h post-hCG; these differences were no longer detectable by 48 h post-hCG. The C/EBP target Gas1 is induced in granulosa cells and is associated with ovulation and luteinization.

Influence of mRNA expression of epiregulin and amphiregulin on outcome of patients with metastatic colorectal cancer treated with 5-FU/LV plus irinotecan or irinotecan plus oxaliplatin as first-line treatment (FIRE 1-trial).

Our aim was to investigate the impact of EREG and AREG mRNA expression (by RT-qPCR) in patients with metastatic colorectal cancer (mCRC). In addition, epidermal growth factor receptor (EGFR) expression (by immunohistochemistry) as well as RAS-and PIK3CA-mutations (by pyrosequencing) were assessed. Tumors of 208 mCRC patients receiving 5-fluorouracil/leucovorin plus irinotecan (FUFIRI) or irinotecan plus oxaliplatin (mIROX) within the FIRE-1 trial were analyzed for mutations. Molecular characteristics were correlated with response, progression-free survival (PFS), overall survival (OS). mRNA expression was evaluated using ROC-analysis in 192 tumors (AREG high n = 31 vs. low n = 161; EREG high n = 89 vs. low n = 103). High versus low AREG expression was associated with PFS of 10.0 versus 8.0 months (HR = 0.62, 95% CI: 0.402-0.940, p = 0.03) and OS of 24.6 versus 18.7 months (HR = 0.72, 95% CI: 0.476-1.078, p = 0.11). High versus low EREG expression correlated with prolonged PFS (9.4 vs. 6.8 months, HR = 0.62, 95% CI: 0.460-0.846, p = 0.002) and OS (25.8 vs. 15.5 months, HR = 0.48, 95% CI: 0.351-0.657, p < 0.001). The positive prognostic effect of high EREG expression was confirmed in a multivariate analysis and was neither affected by EGFR expression nor by mutations of RAS- and PIK3CA-genes. EREG expression appears as an independent prognostic marker in patients with mCRC receiving first-line irinotecan-based chemotherapy.

Matrix metalloproteinase inhibition influences aspects of photoperiod stimulated ovarian recrudescence in Siberian hamsters

Blocking matrix metalloproteinase (MMP) activity in vivo with inhibitor GM6001 impedes photostimulated ovarian recrudescence in photoregressed Siberian hamsters. Since direct and indirect effects of MMPs influence a myriad of ovarian functions, we investigated the effect of in vivo MMP inhibition during recrudescence on ovarian mRNA expression of steroidogenic acute regulatory protein (StAR), 3β-hydroxysteroid dehydrogenase (3β-HSD), Cyp19a1 aromatase, epidermal growth factor receptor (EGFR), amphiregulin (Areg), estrogen receptors (Esr1 and Esr2), tissue inhibitors of MMPs (TIMP-1,-2,-3), proliferating cell nuclear antigen (PCNA), vascular endothelial growth factor A (VEGFA), its receptor VEGFR-2, and angiopoietin-2 (Ang-2). Female Siberian hamsters were randomly assigned to one of four photoperiod groups: stimulatory long (LD) or inhibitory short (SD) photoperiods, or transferred from SD to LD for 2weeks (post-transfer, PT). Half of the PT hamsters were injected (ip) daily with GM6001 (PTG). SD exposure reduced ovarian StAR, 3β-HSD, Cyp19a1, Esr1, Esr2, TIMPs 2–3, PCNA, VEGFR-2 and Ang-2 mRNA expression (p<0.05), and 2weeks of photostimulation restored mRNA expression of 3β-HSD and PCNA and increased Areg and VEGFA mRNA expression in the PT group. GM6001 treatment during photostimulation (PTG) increased TIMP-1, -2 and -3 and PCNA mRNA, but inhibited Areg mRNA expression compared to PT. Neither photoperiod nor GM6001 altered EGFR expression. Results of this study suggest that in vivo inhibition of MMP activity by GM6001 may impede ovarian recrudescence, particularly follicular growth, in two ways: (1) directly by partially inhibiting the release of EGFR ligands like Areg, thereby potentially affecting EGFR activation and its downstream pathway, and (2) indirectly by its effect on TIMPs which themselves can affect proliferation, angiogenesis and follicular growth.

Estimation of safety/risk profile of the phytoestrogens 8-prenylnaringenin, 6-(1.1-dimethylallyl)naringenin and naringenin in MCF-7 cells and the rat mammary gland

Evaluation of stimulation of proliferation in female reproductive organs by phytoestrogens of reproductive tract organs is important to estimate their safety/risk profile if used as an alternative to hormone therapy. To assess the proliferative capacity of the flavanones 8-Prenylnaringenin (8-PN), 6-(1.1-dimethylallyl)naringenin (6-DMAN) and Naringenin (Nar) on the mammary gland we compared their estrogenic properties to 17β-Estradiol (E2) in three independent assays. We performed competitive estrogen receptor (ER) ligand binding assays, MCF-7 proliferation studies and a three day exposure experiment in young adult ovariectomized female rats. 8-PN displayed a high relative binding affinity for both ERs with a preference for ERα (19.46%) compared to ERβ (6.5%). Among the tested flavanones 8-PN had the strongest mitotic effect on MCF-7 cells. In vivo 8-PN increased the number of terminal end buds in the mammary gland and the protein expression of the proliferation markers Ki-67 and PCNA in epithelial ductal cells and further led to an increase of Amphiregulin (Areg) mRNA expression, endpoints correlated to mammary gland proliferation. 6-DMAN also possesses a higher relative binding affinity for the ERα than for the ERβ and stimulated the cell proliferation of MCF-7 cells. It enhanced Ki-67 protein expression and β-Casein mRNA expression in vivo. Nar displayed the weakest estrogenic properties and was the only tested compound with a higher affinity for the ERβ than the ERα. It displayed proliferative effects on MCF-7 cells and slightly induced Ki-67 protein expression in vivo.

MiR-34a suppresses amphiregulin and tumor metastatic potential of head and neck squamous cell carcinoma (HNSCC).

MiR-34a is a well-known tumor metastasis inhibitor, but only a few target genes involved in metastasis have been identified. In HNSCC, the role of miR-34a in metastasis has not been fully elaborated, and the target gene of miR-34a is still blind. Here we addressed that, the relative lower expression of miR-34a is associated with HNSCC lymphatic metastasis. HNSCC metastasis was found to be strongly suppressed in vitro and in vivo by over-expressing miR-34a. In order to screen the possible target genes of miR-34a in HNSCC, a microarray-based differential mRNA profiling mediated by miR-34a over-expression was performed, and AREG was identified as a pivotal target. We demonstrated that the mRNA and protein levels of AREG were greatly reduced when forcing miR-34a expression. The correlation between AREG mRNA levels and HNSCC metastatic phenotype was also significant in HNSCC tissues (p < 0.01). Moreover, the results of luciferase assay provided the further evidence that miR-34a degraded AREG mRNA through targeting the 3'-UTR site. Restoration of AREG expression partially rescued miR-34a-mediated cell invasion defects in vivo and in vitro. Additionally, Over-expressing miR-34a greatly reduced EGFR and uPA, which were reversed by re-expression of AREG. Taken together, these findings indicate that miR-34a targets AREG, and is essential in inhibition of HNSCC metastasis.

Abstract 625: Proliferative effects of the phytoestrogens 8-Prenylnaringenin, 6-(1.1-dimethylallyl)naringenin and Naringenin in MCF-7 cells and the rat mammary gland

Abstract Evaluation of stimulation of proliferation in female reproductive organs by phytoestrogens of reproductive tract organs is important to estimate their safety/risk profile if used as an alternative to hormone therapy. To assess the proliferative capacity of the flavanones 8-Prenylnaringenin (8-PN), 6-(1.1-dimethylallyl)naringenin (6-DMAN) and Naringenin (Nar) on the mammary gland we compared their estrogenic properties to 17β-Estradiol (E2) in three independent assays. We performed competitive estrogen receptor (ER) ligand binding assays, MCF-7 proliferation studies and a three day exposure experiment in young adult ovariectomized female rats. 8-PN displayed a high relative binding affinity for both ERs with a preference for ERα (19.46 %) compared to ERβ (6.5 %). Among the tested flavanones 8-PN had the strongest mitotic effect on MCF-7 cells. In vivo 8-PN increased the number of terminal end buds (TEB) in the mammary gland and the protein expression of the proliferation markers Ki-67 and PCNA in epithelial ductal cells and further led to an increase of Amphiregulin (Areg) mRNA expression, endpoints correlated to mammary gland proliferation. 6-DMAN also possesses a higher relative binding affinity for the ERα than for the ERβ (1.46 %, 0.84 %) and stimulated the cell proliferation of MCF-7 cells. It enhanced Ki-67 protein expression and β-Casein mRNA expression in vivo. Nar displayed the weakest estrogenic properties and was the only tested compound with a higher affinity for the ERβ than the ERα (0.5 %, 0.08 %). It displayed proliferative effects on MCF-7 cells and slightly induced Ki-67 protein expression in vivo. Citation Format: Janina Helle, Oliver Zierau, Kristin Kräker, Annekathrin Keiler, Günter Vollmer, JoEllen Welsh, Georg Kretzschmar. Proliferative effects of the phytoestrogens 8-Prenylnaringenin, 6-(1.1-dimethylallyl)naringenin and Naringenin in MCF-7 cells and the rat mammary gland. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 625. doi:10.1158/1538-7445.AM2014-625

Expression of amphiregulin in various models of polycystic kidney disease (690.1)

In polycystic kidney disease (PKD) there is the continual expansion of cysts throughout the time course of this disease. The mechanisms that control the rate of cystic growth are not fully known. In PKD, the epidermal growth factor (EGF) receptor is mislocalized to the apical membrane of epithelial cells that line cystic structures. The findings that cystic fluid contains various hormones and signaling molecules further suggests that growth factors may play a role in regulation of cystic growth. The purpose of this study was to determine which EGF receptor dependent growth factors are elevated in PKD. Growth factors from human cystic fluid and mouse models of PKD were measured with an Elisa assay and RT‐PCR. Amphiregulin (Areg), epiregulin (Ereg), transforming growth factor alpha (TGFα), heparin binding EGF (HBegf) and egf were measured. In the IFT88 knockout mouse model where cilia are deleted, there was a four‐fold increase in Areg mRNA expression in mice that lacked cilia compared to mice with cilia. All other growth factors were the same between cilia minus versus cilia plus mice. In mice with a knockout of polycystin 2 there was a 5 fold increase in only Areg compared to mice with polycystin 2. In mice that lacked polycystin 1 there was 3 fold increase in Areg and an 8 fold increase in Ereg. Other growth factors were unchanged by the presence or absence of these proteins that cause autosomal dominant PKD (ADPKD). In fluid collected from individual cysts from human ADPKD kidneys we found that Areg concentration varied between 49 to 250 pg\ml and also varied between cysts from the same kidney. These results suggest that Areg and possibility Ereg may play a role in cystogenesis in PKD.Grant Funding Source: P30DK074038 and VA Merit