Dephosphorylation Of Sp1 Research Articles

MiR-181b modulates EGFR-dependent VCAM-1 expression and monocyte adhesion in glioblastoma

Tumor-associated macrophages (TAMs) originate as circulating monocytes, and are recruited to gliomas, where they facilitate tumor growth and migration. Understanding the interaction between TAM and cancer cells may identify therapeutic targets for glioblastoma multiforme (GBM). Vascular cell adhesion molecule-1 (VCAM-1) is a cytokine-induced adhesion molecule expressed on the surface of cancer cells, which is involved in interactions with immune cells. Analysis of the glioma patient database and tissue immunohistochemistry showed that VCAM-1 expression correlated with the clinico-pathological grade of gliomas. Here, we found that VCAM-1 expression correlated positively with monocyte adhesion to GBM, and knockdown of VCAM-1 abolished the enhancement of monocyte adhesion. Importantly, upregulation of VCAM-1 is dependent on epidermal-growth-factor-receptor (EGFR) expression, and inhibition of EGFR effectively reduced VCAM-1 expression and monocyte adhesion activity. Moreover, GBM possessing higher EGFR levels (U251 cells) had higher VCAM-1 levels compared to GBMs with lower levels of EGFR (GL261 cells). Using two- and three-dimensional cultures, we found that monocyte adhesion to GBM occurs via integrin α4β1, which promotes tumor growth and invasion activity. Increased proliferation and tumor necrosis factor-α and IFN-γ levels were also observed in the adherent monocytes. Using a genetic modification approach, we demonstrated that VCAM-1 expression and monocyte adhesion were regulated by the miR-181 family, and lower levels of miR-181b correlated with high-grade glioma patients. Our results also demonstrated that miR-181b/protein phosphatase 2A-modulated SP-1 de-phosphorylation, which mediated the EGFR-dependent VCAM-1 expression and monocyte adhesion to GBM. We also found that the EGFR-dependent VCAM-1 expression is mediated by the p38/STAT3 signaling pathway. Our study suggested that VCAM-1 is a critical modulator of EGFR-dependent interaction of monocytes with GBM, which raises the possibility of developing effective and improved therapies for GBM.

Physiological TLR5 expression in the intestine is regulated by differential DNA binding of Sp1/Sp3 through simultaneous Sp1 dephosphorylation and Sp3 phosphorylation by two different PKC isoforms.

Toll-like receptor 5 (TLR5) expression in the intestinal epithelial cells (IECs) is critical to maintain health, as underscored by multiple intestinal and extra-intestinal diseases in mice genetically engineered for IEC-specific TLR5 knockout. A gradient of expression exists in the colonic epithelial cells from the cecum to the distal colon. Intriguingly, an identical gradient for the dietary metabolite, butyrate also exists in the luminal contents. However, both being critical for intestinal homeostasis and immune response, no studies examined the role of butyrate in the regulation of TLR5 expression. We showed that butyrate transcriptionally upregulates TLR5 in the IECs and augments flagellin-induced immune responses. Both basal and butyrate-induced transcription is regulated by differential binding of Sp-family transcription factors to the GC-box sequences over the TLR5 promoter. Butyrate activates two different protein kinase C isoforms to dephosphorylate/acetylate Sp1 by serine/threonine phosphatases and phosphorylate Sp3 by ERK-MAPK, respectively. This resulted in Sp1 displacement from the promoter and binding of Sp3 to it, leading to p300 recruitment and histone acetylation, activating transcription. This is the first study addressing the mechanisms of physiological TLR5 expression in the intestine. Additionally, a novel insight is gained into Sp1/Sp3-mediated gene regulation that may apply to other genes.

Dephosphorylation of Sp1 at Ser-59 by Protein Phosphatase 2A (PP2A) is required for induction of CYP1A1 transcription after treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin or omeprazole

The aryl hydrocarbon receptor (AhR) is a transcription factor that is activated by either 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or omeprazole (OP). Activated AhR can induce CYP1A1 transcription by binding to the xenobiotic responsive element (XRE). However, the mechanism of activation of the CYP1A1 promoter region is poorly understood. Previous reports showed that Sp1 could bind to a GC-rich region near the CYP1A1 promoter. This study sought to clarify the function of Sp1 in CYP1A1 transcription. Phosphorylation of Sp1 at Ser-59 (pSer-59) was previously reported to be closely related to transcriptional regulation. We used a site-specific phospho-antibody to show that treatment with TCDD or OP drastically reduced the level of pSer-59 in Sp1 from HepG2 cells. This reduction was too much, we hypothesized that the reduced phosphorylation level resulted from activation of phosphatase activity. Given that pSer-59 is dephosphorylated by PP2A, we examined the effect of a PP2A inhibitor, okadaic acid (OA), on pSer-59 and transcription of CYP1A1. The results showed that OA blocked dephosphorylation of Ser-59 and drastically inhibited transcription of CYP1A1. Similar results were obtained after knockdown of PP2A. Treatment with OA had no effect on the expression of AhR, its nuclear translocation, or its ability to bind to the XRE. Furthermore, dephosphorylation of Sp1 at Ser-59 was not affected by knockdown of AhR. These results indicate that the signals from TCDD or OP caused PP2A-mediated dephosphorylation of Sp1 at Ser-59 and induced CYP1A1 transcription. This signaling pathway was independent of the AhR-mediated pathway.

Phosphorylation by CDK1 and JNK1 regulate the DNA binding activity of transcription factor Sp1 during mitosis

The transcription factor Sp1 is ubiquitously expressed in different cells and thereby regulates the expression of genes involved in many cellular processes. This study reveals that Sp1 was phosphorylated at threonine 739 by JNK and CDK1 and not co‐localized with the chromatin at the beginning of mitotic stage. EMSA, DNA affinity precipitation assay, and ChIP assay were performed and revealed that during mitosis the DNA‐binding ability of phosphorylated Sp1was decrease as well as dephosphorylation of Sp1 by phosphatase restored the DNA binding affinity. In addition, the phosphorylation of Sp1 during mitotic stage also suppressed Sp1 to interact with p300, Brg1, and RNA polII and reduced the transcriptional activity of Sp1 target genes. Moreover, in late mitosis, the Pin1 interacted with the high phosphorylation of Sp1 and recruited the PP2A to dephosphorylate Sp1. In Pin1 knockout MEF cells, there was a delay in the relocalization of Sp1 to the nucleus during telophase. Taken together, our results indicated that Sp1 interacts with JNK and CDK1, followed by phosphorylation of Sp1 at early mitosis. Sp1, then, is disassociated from DNA and loses interaction with co‐activator. In late mitosis, Pin1 and PP2A are recruited by phosphorylation of Sp1, leading to the enhancement of the DNA binding‐ability and protein interactional function of Sp1.

Artemisinin Blocks Prostate Cancer Growth and Cell Cycle Progression by Disrupting Sp1 Interactions with the Cyclin-dependent Kinase-4 (CDK4) Promoter and Inhibiting CDK4 Gene Expression

Artemisinin, a naturally occurring component of Artemisia annua, or sweet wormwood, is a potent anti-malaria compound that has recently been shown to have anti-proliferative effects on a number of human cancer cell types, although little is know about the molecular mechanisms of this response. We have observed that artemisinin treatment triggers a stringent G1 cell cycle arrest of LNCaP (lymph node carcinoma of the prostate) human prostate cancer cells that is accompanied by a rapid down-regulation of CDK2 and CDK4 protein and transcript levels. Transient transfection with promoter-linked luciferase reporter plasmids revealed that artemisinin strongly inhibits CDK2 and CDK4 promoter activity. Deletion analysis of the CDK4 promoter revealed a 231-bp artemisinin-responsive region between -1737 and -1506. Site-specific mutations revealed that the Sp1 site at -1531 was necessary for artemisinin responsiveness in the context of the CDK4 promoter. DNA binding assays as well as chromatin immunoprecipitation assays demonstrated that this Sp1-binding site in the CDK4 promoter forms a specific artemisinin-responsive DNA-protein complex that contains the Sp1 transcription factor. Artemisinin reduced phosphorylation of Sp1, and when dephosphorylation of Sp1 was inhibited by treatment of cells with the phosphatase inhibitor okadaic acid, the ability of artemisinin to down-regulate Sp1 interactions with the CDK4 promoter was ablated, rendering the CDK4 promoter unresponsive to artemisinin. Finally, overexpression of Sp1 mostly reversed the artemisinin down-regulation of CDK4 promoter activity and partially reversed the cell cycle arrest. Taken together, our results demonstrate that a key event in the artemisinin anti-proliferative effects in prostate cancer cells is the transcriptional down-regulation of CDK4 expression by disruption of Sp1 interactions with the CDK4 promoter.

PTEN downregulates p75NTR expression by decreasing DNA-binding activity of Sp1

p75NTR is expressed throughout the nervous system and its dysregulation is associated with pathological conditions. We have recently demonstrated a signalling cascade initiated by laminin (LN), which upregulates PTEN and downregulates p75NTR. Here we investigate the mechanism by which PTEN modulates p75NTR. Studies using PTEN mutants show that its protein phosphatase activity directly modulates p75NTR protein expression. Nuclear relocalization of PTEN subsequent to LN stimulation suggests transcriptional control of p75NTR expression, which was confirmed following EMSA and ChIP analysis of Sp1 transcription factor binding activity. LN and PTEN independently decrease the DNA-binding ability of PTEN to the p75NTR promoter. Sp1 regulation of p75NTR occurs via dephosphorylation of Sp1, thus reducing p75NTR transcription and protein expression. This mechanism represents a novel regulatory pathway which controls the expression level of a receptor with broad implications not only for the development of the nervous system but also for progression of pathological conditions.

Butyrate-induced phosphatase regulates VEGF and angiogenesis via Sp1

Sp1 is a ubiquitous transcription factor and master regulator of various eukaryotic gene expression. Better understanding of the role of increased Sp1 levels on angiogenic regulation and the regulatory regions of that transcription factor may act as a useful target in ‘transcriptional therapy’. At the molecular level, butyrate inhibits Sp1-DNA binding activity by promoting Sp1 protein dephosphorylation in EAT cells. It also inhibits Sp1 binding activity and reduces expression of VEGF gene, thereby inhibiting angiogenesis. It was confirmed that butyrate induces expression of a tyrosine phosphatase by RT-PCR, cDNA sequence analysis, protein ESI-MS analysis and protein sequence homology comparison. Thus our result strongly suggests that inhibition of angiogenesis by butyrate involves Sp1 dephosphorylation and down-regulation of VEGF gene expression. Further, butyrate inhibits neoangiogenesis induced by tumor cells and VEGF in peritoneum of EAT bearing mice and rat cornea.

Increased Chromatin Association of Sp1 in Interphase Cells by PP2A-mediated Dephosphorylations

Sp1 dephosphorylation by phosphatase 2A is related to sustained cellular proliferation and is illustrated by an enhanced electrophoretic migration shift. This event occurs concurrently with cell-cycle interphase and increases Sp1 transcriptional activity and in vitro affinity for DNA. We show here that dephosphorylated Sp1 is associated with chromatin more tightly than its phosphorylated counterparts from either resting or mitotic cells. Analysis of the expression of Sp1 point mutants and use of a phospho-specific antibody enabled identification of serine 59 as a major target of PP2A during cell-cycle interphase. Importantly, serine 59 dephosphorylation appeared to up-regulate Sp1 association with chromatin. Various studies suggested that this might occur through the control of the reciprocal O-phosphate/ O-GlcNAc modification of other residues, some of which are likely to belong to the Sp1 C-terminal DNA-binding domain. In addition, we demonstrated by phosphopeptide mapping that threonine 681, which belongs to the latter region, is another target of PP2A, yet unrelated to serine 59. We propose that the coordinated dephosphorylation of several Sp1 residues, a general feature of dividing cells, is a required post-translational mechanism for Sp1-dependent transcription of genes related to cell division.

Human immunodeficiency virus type 1 Tat prevents dephosphorylation of Sp1 by TCF-4 in astrocytes

Previous examination of the effect of TCF-4 on transcription of the human immunodeficiency virus type 1 (HIV-1) promoter in human astrocytic cells found that TCF-4 affects the HIV-1 promoter through the GC-rich domain (nt -80 to nt -68). Here, the physical interaction and a functional consequence of TCF4-Sp1 contact were characterized. It was shown that expression of TCF-4 in U-87 MG (human astrocytic) cells decreased basal and Sp1-mediated transcription of the HIV-1 promoter. Results from a GST pull-down assay, as well as combined immunoprecipitation and Western blot analysis of protein extracts from U-87 MG cells, revealed an interaction of Sp1 with TCF-4. Using in vitro protein chromatography, the region of Sp1 that contacts TCF-4 was mapped to aa 266-350. It was also found that, in cell-free extracts, TCF-4 prevented dsDNA-dependent protein kinase (DNA-PK)-mediated Sp1 phosphorylation. Surprisingly, TCF-4 failed to decrease Sp1-mediated transcription of the HIV-1 long terminal repeat (LTR) and Sp1 phosphorylation in cells expressing HIV-1 Tat. Results from immunoprecipitation/Western blotting demonstrated that TCF-4 lost its ability to interact with Sp1, but not with Tat, in Tat-transfected cells. Taken together, these findings suggest that activity at the HIV-1 promoter is influenced by phosphorylation of Sp1, which is affected by Tat and DNA-PK. Interactions among TCF-4, Sp1 and/or Tat may determine the level of viral gene transcription in human astrocytic cells.

Interleukin-21 Receptor Gene Induction in Human T Cells Is Mediated by T-Cell Receptor-Induced Sp1 Activity

Interleukin-21 (IL-21) plays important roles in regulating the immune response. IL-21 receptor (IL-21R) mRNA is expressed at a low level in human resting T cells but is rapidly induced by mitogenic stimulation. We now investigate the basis for IL21R gene regulation in T cells. We found that the -80 to -20 region critically regulates IL-21R promoter activity and corresponds to a major DNase I-hypersensitive site. Electrophoretic mobility shift assays, DNA affinity chromatography followed by mass spectrometry, and chromatin immunoprecipitation assays revealed that Sp1 binds to this region in vitro and in vivo. Moreover, mutation of the Sp1 motif markedly reduced IL-21R promoter activity, and Sp1 small interfering RNAs effectively diminished IL-21R expression in activated T cells. Interestingly, upon T-cell receptor (TCR) stimulation, T cells increased IL-21R expression and Sp1 protein levels while decreasing Sp1 phosphorylation. Moreover, phosphatase inhibitors that increased phosphorylation of Sp1 diminished IL-21R transcription. These data indicate that TCR-induced IL-21R expression is driven by TCR-mediated augmentation of Sp1 protein levels and may partly depend on the dephosphorylation of Sp1.

Progesterone receptor activates its promoter activity in human endometrial stromal cells

Previous studies have shown that progestin increases the content of progesterone receptor (hPRA and hPRB) and the hPR mRNA during decidualization of human endometrial stromal cells suggesting that endogenous hPR enhances the transcription of the hPR gene. In the present study, we provide evidence that hPR regulates the promoter activity mediated through an active Sp1 site. In stromal cells treated with medroxyprogesterone acetate, the promoter activity was significantly increased when cells were co-transfected with hPR expression vector. Progressive deletion analysis showed that the highest activity was derived from the promoter region between −55 and +31 bp. Transactivation by hPR was dose dependent. The capacity of hPRA was stronger than that of hPRB. The ligand binding domain, but not DNA binding domain of the hPR was required for the transactivation. The proximal promoter region lacks a canonical progesterone response element. Instead, an active Sp1 site (−49 to −43 bp) has been confirmed. Mutation of the Sp1 site eliminated the effect of hPR activation. The promoter activity was increased by over expression of Sp1, whereas Sp3 had no effect. Electrophoretic mobility shift assay showed that the promoter region between −55 and +31 bp bound to Sp1 family proteins, Sp1 (C2 complex) and Sp3 (C1 and C3 complexes) identified by antibodies to Sp1 and Sp3. Sp1 complex formed by extracts of stromal cells was less intense than that formed by progestin-decidualized stromal cells. Sp1/DNA binding was enhanced when stromal cell extracts were incubated with calf intestine alkaline phosphatase (CIP) suggesting that dephosphorylation of Sp1 enhances the DNA binding. Addition of protein kinase inhibitor, H-89 or H-7, enhanced the hPR stimulated promoter activity. Western blot analysis showed that endometrial stromal/decidual cell extracts contained a wide band of Sp1 spanning from ∼105 to 96 kDa and was resolved into one band at 96 kDa by CIP. Decidual cell extracts are abundant with the 96 kDa Sp1. In addition, the 96 kDa Sp1 was co-precipitated with ligand-activated hPRA or hPRB in the decidual cell nuclear extracts. These data suggest that dephosphorylated Sp1, abundant in decidual cells, enhances the binding to both DNA and hPR resulting in a robust increase of the hPR promoter activity.

Adaptation and maladaptation of the heart in diabetes: Part II: potential mechanisms.

The prevailing concept of the heart’s response to changes in its environment is a complex network of inter-connecting signal transduction cascades.1 In such a scheme, the focus is on communication of various cell surface receptors, heterotrimeric G-proteins, protein kinases, and transcription factors.2–4⇓⇓ Diabetes is a disorder of metabolic dysregulation. At first glance it appears that metabolism and the metabolic consequences of diabetes do not fit into this signal-response coupling scheme. Two questions arise. First, is metabolism simply an “effect” rather than a “cause” of adaptation? Second, is metabolism only a by-product of signal transduction-induced adaptation, allowing equilibrium (and therefore maintenance of function) in the presence of the other adaptational responses? An alternative is to take a new, less restricted view of metabolism. Beyond its stereotypical function as a provider of ATP, alterations in metabolic flux within the cell create essential signals for the adaptation of the heart to situations such as diabetes. This concept is novel for the heart, but has already been considered in the liver. Like the phosphorylation events occurring in signal transduction cascades, changes in metabolic flux are extremely rapid. For example, translocation of GLUT4 to the cell surface in response to insulin occurs within a second.5 We have previously found that increases or decreases in workload also change metabolic fluxes in seconds.6,7⇓ Therefore, changes in metabolites are rapid enough to allow them to act as signaling molecules. Many of these acute changes in metabolic flux are brought about by the same signal transduction cascades believed to be involved in the adaptation of the heart to changes in its environment. Phosphatidylinositol 3-kinase, Ca2+, and protein kinase C, all of which play a role in cardiac adaptation, regulate metabolism in the heart.8,9⇓ Metabolic signals therefore provide a …

Sp1 Transcriptional Activity Is Up-regulated by Phosphatase 2A in Dividing T Lymphocytes

We have followed Sp1 expression in primary human T lymphocytes induced, via CD2 plus CD28 costimulation, to sustained proliferation and subsequent return to quiescence. Binding of Sp1 to wheat germ agglutinin lectin was not modified following activation, indicating that the overall glycosylation of the protein was unchanged. Sp1 underwent, instead, a major dephosphorylation that correlated with cyclin A expression and, thus, with cell cycle progression. A similar change was observed in T cells that re-entered cell cycle following secondary interleukin-2 stimulation, as well as in serum-induced proliferating NIH/3T3 fibroblasts. Phosphatase 2A (PP2A) appears involved because 1) treatment of dividing cells with okadaic acid or cantharidin inhibited Sp1 dephosphorylation and 2) PP2A dephosphorylated Sp1 in vitro and strongly interacted with Sp1 in vivo. Sp1 dephosphorylation is likely to increase its transcriptional activity because PP2A overexpression potentiated Sp1 site-driven chloramphenicol acetyltransferase expression in dividing Kit225 T cells and okadaic acid reversed this effect. This increase might be mediated by a stronger affinity of dephosphorylated Sp1 for DNA, as illustrated by the reduced DNA occupancy by hyperphosphorylated Sp factors from cantharidin- or nocodazole-treated cells. Finally, Sp1 dephosphorylation appears to occur throughout cell cycle except for mitosis, a likely common feature to all cycling cells.

Differential Expression of the Adipocyte Amino Acid Transporter Is Transactivated by SP1 and SP3 during the 3T3-L1 Preadipocyte Differentiation Process

It was identified that a Sp1-like cis-element in the adipocyte amino acid transporter gene (AAAT) promoter is the major cis-motif for its induced expression during the 3T3-L1 preadipocyte differentiation process. Electrophoretic mobility shift analysis of this cis DNA element showed that the transcription factors binding to this sequence were Sp1 and Sp3. Protein analysis of Sp1 and Sp3 in nuclear extracts from 3T3-L1 cells at various differentiation stages indicated that these two transcription factors existed in noninduced 3T3-L1 preadipocytes, but they did not bind to the AAAT promoter element with high affinity. They were activated after differentiation induction. It was further demonstrated that dephosphorylation of Sp1 increased its binding affinity to this inducible AAAT promoter element.

Sp1 and Sp3 Regulate Transcriptional Activity of the Facilitative Glucose Transporter Isoform-3 Gene in Mammalian Neuroblasts and Trophoblasts

The murine facilitative glucose transporter isoform 3 (Glut 3) is developmentally regulated and is predominantly expressed in neurons and trophoblasts. Employing the primer extension and RNase protection assays, the transcription start site (denoted as +1) of the murine Glut 3 gene was localized to 305 base pairs (bp) 5' to the ATG translation start codon. Transient transfection assays in N2A, H19-7 neuroblasts, and HRP.1 trophoblasts using sequential 5'-deletions of the murine Glut 3-luciferase fusion gene indicated that the -203 to +237 bp region with reference to the transcriptional start site contained promoter activity. Repressor function was limited to the -137 to -130 bp region within the transcriptional activation domain. The nuclear factors Sp1 and Sp3 bound this GC-rich region in N2A, H19-7, and HRP.1 cells. Dephosphorylation of Sp1 was essential for Glut 3 DNA binding. The related Sp3 protein also bound this same region of mouse Glut 3 in all three cell lines. Mutations of the Sp1-binding site employed in transient transfection and mobility shift assays confirmed the nature of the DNA-binding proteins, while supershift assays with anti-Sp1 and anti-Sp3 IgGs characterized the differences in the two DNA-binding proteins. Co-transfection of the Glut 3-luciferase fusion gene with or without mutations of the Sp1-binding site along with the Sp1 or Sp3 expression vectors in Drosophila SL2 cells confirmed a reciprocal effect, with Sp1 suppressing and Sp3 activating Glut 3 gene transcription.

Glucose regulates the promoter activity of aldolase A and pyruvate kinase M 2 via dephosphorylation of Sp1

Proliferating cells and tumour cells maintain a high glycolytic rate even under aerobic conditions. FTO2B cells, a rat hepatoma cell line, show high activities of glycolytic enzymes. Within a culture period of 48 h the cell number increases 5-fold. Replacement of glucose by pyruvate in the culture medium lowers glycolytic enzyme activity and prevents proliferation. Transfection assays revealed that glucose deprivation dramatically decreases the transcriptional activities of the Sp1-dependent aldolase and pyruvate kinase promoters leading to reduced reporter gene expression. Sp1 binding activity is also inhibited by ocadaic acid, an inhibitor of protein phosphatase 1. Western blot analyses with nuclear extracts from FTO2B cells cultured in the presence or absence of glucose revealed differences in the phosphorylation state of Sp1. From these results we conclude that glucose increases the amount of the dephosphorylated form of Sp1 which has a higher DNA binding activity. As a consequence gene expression of the glycolytic enzymes is increased which is a prerequisite for cell proliferation.

Protein Kinase CK2 Down-Regulates Glucose-Activated Expression of the Acetyl-CoA Carboxylase Gene

It has been suggested that, in pancreatic β-cells, acetyl-CoA carboxylase (ACC) is a key enzyme in glucose signal transduction leading to glucose-induced insulin secretion. The PII promoter is the only active promoter for the ACC gene in the β-cell. Here we report that, in the pancreatic β-cell, high glucose levels (above 20 mm) activate Sp1 binding to the glucose response element of the PII promoter, which leads to a dose-dependent increase in PII transcription. The expression of a gene coding protein kinase CK2 (CK2) α subunit, or the presence of okadaic acid (a serine/threonine protein phosphatase inhibitor), partially blocks the glucose activation of PII transcription. The inhibitory effect of CK2 α, or okadaic acid, was not observed in the absence of glucose or at low glucose concentrations. Phosphorylation of Sp1 by CK2 α leads to the inactivation of Sp1 binding to PII. Dephosphorylation of the phosphorylated Sp1 by protein phosphatase 1 (PP1) activates the binding of Sp1 to PII. Inhibition of PP1-catalyzed Sp1 dephosphorylation by okadaic acid, or PP1 specific inhibitor 2, decreases Sp1 binding to PII. These results suggest that the phosphorylation/dephosphorylation of Sp1 by CK2/PP1 may be the underlying mechanism by which the expression of the PII promoter of ACC is controlled in the process of glucose-mediated insulin secretion in pancreatic β-cells.

Dephosphorylation of Sp1 by Protein Phosphatase 1 Is Involved in the Glucose-mediated Activation of the Acetyl-CoA Carboxylase Gene

When mouse 30A5 preadipocytes are exposed to high glucose concentrations, acetyl-CoA carboxylase is induced through glucose activation of promoter II of the acetyl-CoA carboxylase gene. Glucose treatment of the cells increases Sp1 binding to two GC-rich glucose response elements in promoter II. We have investigated the mechanism by which glucose increases Sp1 binding and transactivation of promoter II in 30A5 cells. DNA mobility shift assays have shown that nuclear extracts from glucose-treated cells exhibit increased Sp1 binding activity. This increase in the binding activity is not due to glucose-mediated changes in the amount of Sp1 in the nucleus but to an increase in the activity that modifies Sp1 so that it binds more effectively to the promoter sequence. This Sp1 modifying activity is inhibited by okadaic acid and phosphatase inhibitor 2, and has a molecular mass of 38-42 kDa. The catalytic subunit of type 1 protein phosphatase, whose molecular mass is 38 kDa, also increased the ability of Sp1 to bind to promoter II. Treatment of nuclear extract with antibodies against the catalytic subunit partially suppressed the nuclear activity for Sp1 activation. From these results, we conclude that the Sp1 transcription factor exhibits enhanced binding to promoter II and transcriptional activation is the result of glucose-induced dephosphorylation by type 1 phosphatase.