Cyclic Nucleotide Regulation of Na+/Glucose Cotransporter (SGLT1) mRNA Stability

Abstract

Expression of the Na؉ -coupled glucose cotransporter SGLT1 is regulated post-transcriptionally at the level of mRNA stability. We have previously demonstrated that cAMP-dependent stabilization of the SGLT1 message was correlated with the protein phosphorylationdependent binding of cytoplasmic proteins to a uridinerich sequence (URE) in the 3-untranslated region (UTR). In the present study, the regulatory role of the URE was demonstrated by inserting it into the 3-UTR of a ␤-globin reporter minigene under the control of the tetracycline-regulated promoter. The resultant chimeric globin/SGLT1 mRNA expressed after transfection into LLC-PK1 cells exhibited a decreased half-life compared with the ␤-globin control, indicating that the URE serves a destabilizing function. Activation of protein kinase A stabilized the chimeric message but not the ␤-globin control, indicating the presence of a regulatory stabilizing sequence within the URE. A 38-kDa nucleocytoplasmic protein was identified that recognized a 12-nucleotide binding site within the URE. A mutation in this binding site that prevented protein binding assayed in vitro by UV cross-linking also prevented protein kinase Adependent stabilization of the chimeric message assayed in vivo. These findings identify the interaction between a 38-kDa nucleocytoplasmic protein and a regulatory uridinerich sequence in the 3-UTR as critical for cAMP-mediated SGLT1 message stabilization.Glucose is the major carbon and energy source for eukaryotic cells. Transport of glucose into mammalian cells is the ratelimiting step for its utilization and accordingly is a highly regulated process. Maintenance of a constant blood glucose level is essential for cellular homeostasis. In kidney, glucose is reabsorbed from the urinary filtrate by the action of several types of glucose transporters arranged in series along the proximal tubule. These function together in polarized epithelial cells to mediate transepithelial transport of glucose. SGLT 1 (Na ϩ /glucose) cotransporters in the apical membrane catalyze active glucose transport into the cell driven by the Na ϩ gradient (1). Glucose diffuses passively out of the cell into the circulation via basolateral GLUT facilitative transporters (2). High affinity glucose transporters SGLT1 and GLUT1 located in late proximal tubule scavenge the remainder of filtered glucose not reabsorbed in early proximal tubule (3). SGLT1 is also expressed in small intestine where it mediates absorption of dietary glucose and galactose (3, 4).Most of our current information concerning the regulation of SGLT1 expression has been obtained from studies using the polarized epithelial cell line LLC-PK 1 , derived from porcine kidney (5). SGLT1 is expressed in this cell line as shown by cDNA cloning and Northern blot analysis (6) and is highly regulated by the cell growth and differentiation state (5,7,8). Protein kinase A (PKA) and protein kinase C (PKC) exert opposing effects on SGLT1 mRNA stability and steady-state levels in confluent cultures. Activation of PKA resu...

Nutritional Control of mRNA Stability Is Mediated by a Conserved AU-rich Element That Binds the Cytoplasmic Shuttling Protein HuR

“…The proteins in these labeled bands are slightly smaller than these sizes because each species contains crosslinked RNA. The 40-kDa species is the size expected for labeled HuR, consistent with results obtained by others (24,25). It is interesting that this species is present at similar levels in nuclear and cytoplasmic extracts, even though HuR is present at much higher levels in nuclear fractions (Fig.…”

“…It is possible that covalent modification of HuR or another protein in the complex could affect the specificity or affinity of one of the proteins. In fact, it has been shown that cAMP stimulates the formation of a HuR-containing complex on the SGLT1 mRNA by a mechanism that involves protein phosphorylation (24,25,28). In addition, it is possible that amino acid starvation causes changes in the levels of another protein that binds to the cat-1 mRNA along with HuR.…”

The Plasma Membrane-associated Protein RS1 Decreases Transcription of the Transporter SGLT1 in Confluent LLC-PK1 Cells

“…They identified a correlation between the increase of SGLT1 mRNA during confluence with an increase of the intracellular cAMP and showed that SGLT1 mRNA was increased by cAMP-elevating agents (22). Furthermore, it was revealed that the cAMP-dependent increase of SGLT1 mRNA relied on a decelerated message degradation that was mediated by binding of a cytosolic protein to a defined 3Ј region of mRNA (21,25). In addition, it was found that the decrease of SGLT1 mRNA during de-differentiation can be blocked or accelerated by inhibition or stimulation of protein kinase C, respectively (19).…”

“…In contrast, the transcriptional regulation of SGLT1 is known to occur more slowly. The expression of SGLT1 is altered via changes of intracellular mRNA concentrations that were reported to be mediated by factors either directly affecting the transcription (11) or the stability of the mRNA (21,25). When LLC-PK 1 cells grown in 5 mM Dglucose differentiate during confluence, the cytosolic concentration of SGLT1 mRNA increases.…”

“…After confluence, protein kinase C is down-regulated, whereas protein kinase A is up-regulated, and the level of SGLT1 mRNA is largely increased (19,22,24). One of the effects of protein kinase A is to enhance message stability, mediated by a specific RNA-binding protein (20,21,25).…”