The protein phosphatase calcineurin determines basal parathyroid hormone gene expression

Calcium and phosphate regulate PTH mRNA stability through differences in binding of parathyroid (PT) proteins to a minimal 63-nucleotide (nt) cis-acting instability element in its 3'-untranslated region. One of these proteins is adenosine-uridine-rich binding factor (AUF1), whose levels are not regulated in PT extracts from rats fed the different diets. However, two-dimensional gels showed posttranslational modification of AUF1 that included phosphorylation. There is no PT cell line, but in HEK 293 cells the 63-nt element is recognized as an instability element, and RNA interference for AUF1 decreased human PTH secretion in cotransfection experiments. Stably transfected cells with a chimeric GH gene containing the PTH 63-nt cis-acting element were used to study the signal transduction pathway that regulates AUF1 modification and chimeric gene mRNA stability. Cyclosporine A, the calcineurin inhibitor, regulated AUF1 posttranslationally, and this correlated with an increase in the stability of GH-PTH 63-nt mRNA but not of the control GH mRNA. Mice with genetic deletion of the calcineurin Abeta gene had markedly increased PTH mRNA levels that were still regulated by low calcium and phosphorus diets. Therefore, calcineurin regulates AUF1 posttranslationally in vitro and PTH gene expression in vivo but still allows its physiological regulation by calcium and phosphate.     

Identification of a novel AU-Rich element in the 3' untranslated region of epidermal growth factor receptor mRNA that is the target for regulated RNA-binding proteins

The epidermal growth factor receptor (EGF-R) plays an important role in the growth and progression of estrogen receptor-negative human breast cancers. EGF binds with high affinity to the EGF-R and activates a variety of second messenger pathways that affect cellular proliferation. However, the underlying mechanisms involved in the regulation of EGF-R expression in breast cancer cells are yet to be described. Here we show that the EGF-induced upregulation of EGF-R mRNA in two human breast cancer cell lines that overexpress EGF-R (MDA-MB-468 and BT-20) is accompanied by stabilization (>2-fold) of EGF-R mRNA. Transient transfections using a luciferase reporter identified a novel EGF-regulated approximately 260-nucleotide (nt) cis-acting element in the 3' untranslated region (3'-UTR) of EGF-R mRNA. This cis element contains two distinct AU-rich sequences (~75 nt), EGF-R1A with two AUUUA pentamers and EGF-R2A with two AUUUUUA extended pentamers. Each independently regulated the mRNA stability of the heterologous reporter. Analysis of mutants of the EGF-R2A AU-rich sequence demonstrated a role for the 3' extended pentamer in regulating basal turnover. RNA gel shift analysis identified cytoplasmic proteins (~55 to 80 kDa) from breast cancer cells that bound specifically to the EGF-R1A and EGF-R2A cis-acting elements and whose binding activity was rapidly downregulated by EGF and phorbol esters. RNA gel shift analysis of EGF-R2A mutants identified a role for the 3' extended AU pentamer, but not the 5' extended pentamer, in binding proteins. These EGF-R mRNA-binding proteins were present in multiple human breast and prostate cancer cell lines. In summary, these data demonstrate a central role for mRNA stabilization in the control of EGF-R gene expression in breast cancer cells. EGF-R mRNA contains a novel complex AU-rich 260-nt cis-acting destabilizing element in the 3'-UTR that is bound by specific and EGF-regulated trans-acting factors. Furthermore, the 3' extended AU pentamer of EGF-R2A plays a central role in regulating EGF-R mRNA stability and the binding of specific RNA-binding proteins. These findings suggest that regulated RNA-protein interactions involving this novel cis-acting element will be a major determinant of EGF-R mRNA stability.     

Regulation of chicken ccn2 gene by interaction between RNA cis-element and putative trans-factor during differentiation of chondrocytes

CCN2/CTGF is a multifunctional growth factor. Our previous studies have revealed that CCN2 plays important roles in both growth and differentiation of chondrocytes and that the 3'-untranslated region (3'-UTR) of ccn2 mRNA contains a cis-repressive element of gene expression. In the present study, we found that the stability of chicken ccn2 mRNA is regulated in a differentiation stage-dependent manner in chondrocytes. We also found that stimulation by bone morphogenetic protein 2, platelet-derived growth factor, and CCN2 stabilized ccn2 mRNA in proliferating chondrocytes but that it destabilized the mRNA in prehypertrophic-hypertrophic chondrocytes. The results of a reporter gene assay revealed that the minimal repressive cis-element of the 3'-UTR of chicken ccn2 mRNA was located within the area between 100 and 150 bases from the polyadenylation tail. Moreover, the stability of ccn2 mRNA was correlated with the interaction between this cis-element and a putative 40-kDa trans-factor in nuclei and cytoplasm. In fact, the binding between them was prominent in proliferating chondrocytes and attenuated in (pre)hypertrophic chondrocytes. Stimulation by the growth factors repressed the binding in proliferating chondrocytes; however, it enhanced it in (pre)hypertrophic chondrocytes. Therefore, gene expression of ccn2 mRNA during endochondral ossification is properly regulated, at least in part, by changing the stability of the mRNA, which arises from the interaction between the RNA cis-element and putative trans-factor.