Post by Max on Jul 17, 2005 7:51:13 GMT -5
Gene-expression through Sp1
Modulation of Sp1 activity by nuclear receptors is a novel mechanism by which fat-soluble hormones regulate gene expression. RARs/RXRs physically interact with Sp1, potentiate Sp1 binding to the GC box motifs, and thus enhance transactivation of the urokinase promoter, which lacks a canonical RAR-responsive element/RXR-responsive element [1]. Nuclear receptors [retinoic acid receptor (RAR), thyroid hormone receptor (TR), vitamin D(3) receptor, peroxisome-proliferator-activated receptor and retinoic X receptor] induce an electrophoretic mobility increase of Sp1-GC-rich DNA complexes. Concomitantly, binding of Sp1 to the GC-box is enhanced [2]. In Ehrlich tumor cells, Sp1-DNA binding was inhibited by phosphatase exposure, demonstrating that phosphorylation of Sp1 is critical for its DNA binding capacity. Immunoprecipitation experiments revealed that Sp1 is mostly phosphorylated on serine residues [3, 7] with less than 5% on threonine and none on tyrosine residues [7].
(Ro)accutane induced significantly decreases of phosphorylation of Sp1 through multiple pathways, leading to a decreased transcription
A list of kinases and phosphatases are known to be involved in Sp1 phosphorylation: DNA-PK, CDK2, ERK, PKC, PI3 kinase, PP1, PP2a and more, depending on cell line and function [7]. Experimental observations have found (Ro)accutane induced significant inhibition of at least three of these, however, it is here suggested that (Ro)accutane can inhibit ERK and possibly even PP1, PP2a and DNA-PK as well:
1) Inhibition of CDK2
(Ro)accutane is found to inhibit multiple of these kinaseses and phosphatases: In LCLs, RA-induced LCL accumulation in the G0/G1 phases correlated with the loss of the catalytic activity of all three G1-associated CDKs (CDK2, CDK4 and CDK6) and with increased levels of underphosphorylated pRb and, in some LCLs, p130. LCLs arrested in G0/G1 by RA also showed a significant decrease in the protein levels of cyclins D2, D3 and A, together with a reduction in the amount of cyclin D associated with CDK4 and CDK6, probably accounting for the inhibition of the relative kinase activity [5]. Cyclin-dependent kinases (CDKs) is essential for cell cycle transversal. CDKs mediate phosphorylation of Sp1 [6].
2) Inhibition of PKC
Protein kinase C from 10T1/2 cells can be eluted by linear gradient of NaCl in two fractions. Following treatment with 10(-5) M 13-cis-retinoic acid a decrease of total PKC activity was observed [8].
3) Inhibition of PI3K/Akt pathway
In MTSV1-7 breast epithelial cells, CRBP-I inhibits, in a retinoic acid receptor-dependent manner, the PI3K/Akt survival pathway. Inhibition of PI3K/Akt was necessary and sufficient to explain the antitumor effects of CRBP-I and was mediated by decreased p85 regulatory and p110 catalytic subunit heterodimerization. We present evidence consistent with the idea that this effect is due to CRBP-I inhibition of p85 phosphorylation at Y688 [9].
Phosphatidylinositol 3-kinase (PI3K) is a critical signaling node that is regulated in response to the activation of growth factor receptors, G protein-coupled receptors, integrins and other cell signals that impact on cell survival and apoptosis. Class Ia PI3Ks consist of a regulatory (most commonly p85) and a catalytic (p110) subunit and catalyze the phosphorylation of phosphatidylinositol at the 3-hydroxy position of the inositide ring. The formation of phosphatidylinositol 3,4,5-trisphosphate (PIP3) from phosphatidylinositol 4,5-bisphosphate is of particular relevance because PIP3 recruits downstream effectors to the plasma membrane. One such effector is Akt, which is activated by phosphoinositide-dependent kinase 1 which is likewise recruited to PIP3 sites. Akt is a serine−threonine protein kinase that once activated acts to inhibit apoptosis by phosphorylation of several key substrates. CRBP-I markedly inhibits Akt activity in cells in collagen I or in suspension relative to adherent monolayers, suggesting that Akt inhibition is a critical path through which CRBP-I promotes anoikis [9].
4) Inhibition of ERK
In HSC-1 cells, suppression of ERK1/2 and Akt activation is presumed to be involved in the RA-induced suppression of hTERT [10].
PPARgamma ligands
In NSCLS cells, PPARgamma ligands also diminished the phosphorylation of cyclic adenosine monophosphate response element binding protein (CREB), diminished Sp1 nuclear protein expression, and prevented the binding of these transcription factors to CRE and Sp1 sites, respectively, within the Fn promoter. In summary, our results demonstrate that PPARgamma ligands inhibit Fn gene expression in NSCLC cells through PPARgamma-dependent and -independent pathways that affect both CREB and Sp1 [4].
Conclusion
In small doses retinoic acid facilitate Sp1 binding to GC boxes (promoter regions). In toxic, supraphysiological doses retinoic acid is suggested to inhibit binding of Sp1 through decreased phosphorylation of Sp1 with at least two signal pathways involved, probably more. The inhibition of Sp1 phosphorylation is here suggested to span over all cell lines and regions where Sp1 promoter activity is essential.
Modulation of Sp1 activity by nuclear receptors is a novel mechanism by which fat-soluble hormones regulate gene expression. RARs/RXRs physically interact with Sp1, potentiate Sp1 binding to the GC box motifs, and thus enhance transactivation of the urokinase promoter, which lacks a canonical RAR-responsive element/RXR-responsive element [1]. Nuclear receptors [retinoic acid receptor (RAR), thyroid hormone receptor (TR), vitamin D(3) receptor, peroxisome-proliferator-activated receptor and retinoic X receptor] induce an electrophoretic mobility increase of Sp1-GC-rich DNA complexes. Concomitantly, binding of Sp1 to the GC-box is enhanced [2]. In Ehrlich tumor cells, Sp1-DNA binding was inhibited by phosphatase exposure, demonstrating that phosphorylation of Sp1 is critical for its DNA binding capacity. Immunoprecipitation experiments revealed that Sp1 is mostly phosphorylated on serine residues [3, 7] with less than 5% on threonine and none on tyrosine residues [7].
(Ro)accutane induced significantly decreases of phosphorylation of Sp1 through multiple pathways, leading to a decreased transcription
A list of kinases and phosphatases are known to be involved in Sp1 phosphorylation: DNA-PK, CDK2, ERK, PKC, PI3 kinase, PP1, PP2a and more, depending on cell line and function [7]. Experimental observations have found (Ro)accutane induced significant inhibition of at least three of these, however, it is here suggested that (Ro)accutane can inhibit ERK and possibly even PP1, PP2a and DNA-PK as well:
1) Inhibition of CDK2
(Ro)accutane is found to inhibit multiple of these kinaseses and phosphatases: In LCLs, RA-induced LCL accumulation in the G0/G1 phases correlated with the loss of the catalytic activity of all three G1-associated CDKs (CDK2, CDK4 and CDK6) and with increased levels of underphosphorylated pRb and, in some LCLs, p130. LCLs arrested in G0/G1 by RA also showed a significant decrease in the protein levels of cyclins D2, D3 and A, together with a reduction in the amount of cyclin D associated with CDK4 and CDK6, probably accounting for the inhibition of the relative kinase activity [5]. Cyclin-dependent kinases (CDKs) is essential for cell cycle transversal. CDKs mediate phosphorylation of Sp1 [6].
2) Inhibition of PKC
Protein kinase C from 10T1/2 cells can be eluted by linear gradient of NaCl in two fractions. Following treatment with 10(-5) M 13-cis-retinoic acid a decrease of total PKC activity was observed [8].
3) Inhibition of PI3K/Akt pathway
In MTSV1-7 breast epithelial cells, CRBP-I inhibits, in a retinoic acid receptor-dependent manner, the PI3K/Akt survival pathway. Inhibition of PI3K/Akt was necessary and sufficient to explain the antitumor effects of CRBP-I and was mediated by decreased p85 regulatory and p110 catalytic subunit heterodimerization. We present evidence consistent with the idea that this effect is due to CRBP-I inhibition of p85 phosphorylation at Y688 [9].
Phosphatidylinositol 3-kinase (PI3K) is a critical signaling node that is regulated in response to the activation of growth factor receptors, G protein-coupled receptors, integrins and other cell signals that impact on cell survival and apoptosis. Class Ia PI3Ks consist of a regulatory (most commonly p85) and a catalytic (p110) subunit and catalyze the phosphorylation of phosphatidylinositol at the 3-hydroxy position of the inositide ring. The formation of phosphatidylinositol 3,4,5-trisphosphate (PIP3) from phosphatidylinositol 4,5-bisphosphate is of particular relevance because PIP3 recruits downstream effectors to the plasma membrane. One such effector is Akt, which is activated by phosphoinositide-dependent kinase 1 which is likewise recruited to PIP3 sites. Akt is a serine−threonine protein kinase that once activated acts to inhibit apoptosis by phosphorylation of several key substrates. CRBP-I markedly inhibits Akt activity in cells in collagen I or in suspension relative to adherent monolayers, suggesting that Akt inhibition is a critical path through which CRBP-I promotes anoikis [9].
4) Inhibition of ERK
In HSC-1 cells, suppression of ERK1/2 and Akt activation is presumed to be involved in the RA-induced suppression of hTERT [10].
PPARgamma ligands
In NSCLS cells, PPARgamma ligands also diminished the phosphorylation of cyclic adenosine monophosphate response element binding protein (CREB), diminished Sp1 nuclear protein expression, and prevented the binding of these transcription factors to CRE and Sp1 sites, respectively, within the Fn promoter. In summary, our results demonstrate that PPARgamma ligands inhibit Fn gene expression in NSCLC cells through PPARgamma-dependent and -independent pathways that affect both CREB and Sp1 [4].
Conclusion
In small doses retinoic acid facilitate Sp1 binding to GC boxes (promoter regions). In toxic, supraphysiological doses retinoic acid is suggested to inhibit binding of Sp1 through decreased phosphorylation of Sp1 with at least two signal pathways involved, probably more. The inhibition of Sp1 phosphorylation is here suggested to span over all cell lines and regions where Sp1 promoter activity is essential.
