AHCYL1 is mediated by estrogen-induced ERK1/2 MAPK cell signaling and microRNA regulation to effect functional aspects of the avian oviduct

Abstract

S-adenosylhomocysteine hydrolase-like protein 1 (AHCYL1), also known as IP(3) receptor-binding protein released with IP(3) (IRBIT), regulates IP(3)-induced Ca(2+) release into the cytoplasm of cells. AHCYL1 is a critical regulator of early developmental stages in zebrafish, but little is known about the function of AHCYL1 or hormonal regulation of expression of the AHCYL1 gene in avian species. Therefore, we investigated differential expression profiles of the AHCYL1 gene in various adult organs and in oviducts from estrogen-treated chickens. Chicken AHCYL1 encodes for a protein of 540 amino acids that is highly conserved and has considerable homology to mammalian AHCYL1 proteins (>94% identity). AHCYL1 mRNA was expressed abundantly in various organs of chickens. Further, the synthetic estrogen agonist induced AHCYL1 mRNA and protein predominantly in luminal and glandular epithelial cells of the chick oviduct. In addition, estrogen activated AHCYL1 through the ERK1/2 signal transduction cascade and that activated expression of AHCYL1 regulated genes affecting oviduct development in chicks as well as calcium release in epithelial cells of the oviduct. Also, microRNAs, miR-124a, miR-1669, miR-1710 and miR-1782 influenced AHCYL1 expression in vitro via its 3'-UTR which suggests that post-transcriptional events are involved in the regulation of AHCYL1 expression in the chick oviduct. In conclusion, these results indicate that AHCYL1 is a novel estrogen-stimulated gene expressed in epithelial cells of the chicken oviduct that likely affects growth, development and calcium metabolism of the mature oviduct of hens via an estrogen-mediated ERK1/2 MAPK cell signaling pathway.

Figure 1. Expression of AHCYL1 in chickens.

[A and B] Expression of AHCYL1 in various organs of male and female of chickens. Results of RT-PCR analysis using cDNA templates from different organs of male [A] and female [B] chickens with chicken AHCYL1 and chicken GAPDH-specific primers. [C] In situ hybridization analyses of AHCYL1 mRNAs in the chicken oviduct. Cross-sections of the four components of the chicken oviduct (infundibulum, magnum, isthmus and shell gland) were hybridized with antisense or sense chicken AHCYL1 cRNA probes. [D] Immunoreactive AHCYL1 protein in the chicken oviduct. For the IgG control, normal mouse IgG was substituted for the primary antibody. Sections were not counterstained. Legend: LE, luminal epithelium; GE, glandular epithelium; Scale bar represents 100 µm. See Materials and Methods for complete description.

Figure 2. Effect of DES on tissue specific expression of chicken AHCYL1.

Both RT-PCR [A and B] and q-PCR [C and D] analyses were performed using cDNA templates from DES-treated and untreated oviducts. These experiments were conducted in triplicate and normalized to control GAPDH expression. [E] In situ hybridization analyses revealed cell-specific expression of AHCYL1 mRNA in oviducts of DES-treated and untreated chicks. Cross-sections of the four segments of chicken oviduct (infundibulum, magnum, isthmus, and shell gland) treated with DES or vehicle were hybridized with antisense or sense chicken AHCYL1 cRNA probes. [F] Immunoreactive AHCYL1 protein in oviducts of DES-treated and untreated chicks. For the IgG control, normal goat IgG was substituted for the primary antibody. Sections were not counterstained. See Materials and Methods for complete description. Legend: Untreated oviduct, non-treated whole oviduct; DES Treatment, DES treated whole oviduct; LE, luminal epithelium; GE, glandular epithelium; Scale bar represents 100 µm. The asterisks denote statistically significant differences (***P<0.001 and **P<0.01).

Figure 3. DES-induced phosphorylation of ERK1/2, stimulation of AHCYL1 and calcium release in chicken oviduct epithelial cells.

[A and B] Dose-dependent and time-dependent expression of phosphorylated ERK1/2 in DES-treated chicken oviduct epithelial cells. Blots were imaged to calculate normalized values presented in graphs (bottom) by measurements of levels of phosphorylated protein relative to total protein. [C] In the DES-treated (2 µg/ml) and non-treated chicken oviduct cells, AHCYL1 protein levels were investigated to determine time-dependent effects of DES. [D] In chicken oviduct cells treated with DES (2 µg/ml) or both DES and an ERK1/2 inhibitor (U0126) for 24 h, according to results of a preliminary study to optimize time-dependent treatment effects, AHCYL1 protein decreased due to effects of U0126. In [C and D], blots were imaged to calculate the normalized values presented in graphs (bottom) for relative abundance of AHCYL1 protein and alpha-tubulin (TUBA) protein. [E] Immunofluorescence microscopy detected AHCYL1 protein in chicken oviduct epithelial cells treated with DES or both DES and an ERK1/2 inhibitor. AHCYL1 protein was barely detectable in untreated, as well as DES- and ERK1/2 inhibitor-treated cells, but abundant in cytoplasm of DES-treated oviduct epithelial cells. Cell nuclei were stained with DAPI (blue). All images were captured at 40X objective magnification. [F and G] Cells were grown in media with various concentration of DES for 24 h or both DES and an ERK1/2 inhibitor. Then, calcium concentration from the cells was measured. The asterisk denotes a significant effect (***P<0.001, **P<0.01 and *P<0.05). See Materials and Methods for complete description.

Figure 4. AHCYL1 knockdown decreased expression of genes associated with oviduct development and production of egg white proteins.

[A] In the control group (naïve, sham and control siRNA treatment) and AHCYL1 silencing group (dose-dependent manner), AHCYL1 mRNA levels were quantified by RT-PCR and quantitative RT-PCR analyses. [B] In the control group (naïve, sham and control siRNA treatment) and AHCYL1 silencing group (dose-dependent), immunoreactive AHCYL1 protein was quantified by western blotting. [C] The effects of DES treatment (time-dependent manner) on control cells and cells in which AHCYL1 was silenced is shown in Panels D to F. Total RNA isolated from chicken oviduct epithelial cells treated with AHCYL1 siRNA (100nM) affected expression of CTSB, CTSC, CTSS, ERRFI1, PTN, GAL11, ovalbumin, LYZ and LYZ2 mRNAs as determined using quantitative RT-PCR analyses. Legend: CTSB, cathepsin B; CTSC, cathepsin C; CTSS, cathepsin S; ERRFI1, ERBB receptor feedback inhibitor 1; PTN, pleiotrophin, GAL11; gallinacin 11, LYZ; lysozyme. [G] Cells were grown in medium with the absence and presence of DES with siRNAs and then changes in amount of calcium released from the cells was measured. [H] Immunofluorescence microscopy detected AHCYL1 protein in chicken oviduct epithelial cells treated with DES with siRNAs. Cell nuclei were stained with DAPI (blue). All images were captured at 40X objective magnification. The asterisks denote statistically significant differences (***P<0.001, **P<0.01 and *P<0.05). See Materials and Methods for complete description.

Figure 5. In vitro target assay of miR-124a, miR-1602, miR-1612, miR-1669, miR-1710 and miR-1782 on AHCYL1 transcript.

[A] Diagram of miR-124a, miR-1669, miR-1710 and miR-1782 binding sites in AHCYL1 3′-UTR. [B] Expression vector maps for eGFP with AHCYL1 3′UTR and Ds-Red with each miRNA. The 3′-UTR of the AHCYL1 transcript was subcloned between the eGFP gene and the polyA tail to generate the fusion construct of the GFP transcript following the miRNA target 3′-UTR (pcDNA-eGFP-3′UTR) (upper panel) and miRNA expression vector was designed to co-express DsRed and each miRNA (pcDNA-DsRed-miRNA) (lower panel). [C and D] After co-transfection of pcDNA-eGFP-3′UTR for the AHCYL1 transcript and pcDNA-DsRed-miRNA for the miR-124a, miR-1669, miR-1710 and miR-1782, the fluorescence signals of GFP and DsRed were detected using fluorescent microscopy [C] and FACS [D]. [E] q-PCR analyses were performed using cDNA templates from DES-treated and untreated chicken oviducts (mean ± SEM). These experiments were conducted in triplicate and normalized to control U6 snRNA expression. See Materials and Methods for complete description. The asterisks denote statistically significant differences (**P<0.01 and *P<0.05).

Figure 6. Schematic illustrating the current working hypothesis on estrogen-induced ERK1/2 MAPK signaling cascades in chicken oviduct cells.

Evidence from the present study indicates that estrogen stimulates the classical estrogen- and alternative ERK1/2 MAPK signaling pathways. Legend: RTK, receptor tyrosine kinase; RAS, synaptic Ras-GTPase-activating protein; RAF (also known as MAPK3), mitogen-activated protein kinase (MAPK) kinase kinase; MEK (also known as MAPK2), MAPK kinase; ERK1/2, extracellular signal-regulated kinase; ERE, estrogen response element; ER, endoplasmic reticulum.