Peroxisome Proliferator-activated Receptor γ-independent Activation of p38 MAPK by Thiazolidinediones Involves Calcium/Calmodulin-dependent Protein Kinase II and Protein Kinase R

The thiazolidinediones (TZDs) are synthetic peroxisome proliferator-activated receptor γ (PPARγ) ligands that promote increased insulin sensitivity in type II diabetic patients. In addition to their ability to improve glucose homeostasis, TZDs also exert anti-proliferative effects by a mechanism that is unclear. Our laboratory has shown that two TZDs, ciglitazone and troglitazone, rapidly induce calcium-dependent p38 mitogen-activated protein kinase (MAPK) phosphorylation in liver epithelial cells. Here, we further characterize the mechanism responsible for p38 MAPK activation by PPARγ ligands and correlate this with the induction of endoplasmic reticulum (ER) stress. Specifically, we show that TZDs rapidly activate the ER stress-responsive pancreatic eukaryotic initiation factor 2α (eIF2α) kinase or PKR (double-stranded RNA-activated protein kinase)-like endoplasmic reticulum kinase/pancreatic eIF2α kinase, and that activation of these kinases is correlated with subsequent eIF2α phosphorylation. Interestingly, PPARγ ligands not only activated calcium/calmodulin-dependent kinase II (CaMKII) 2-fold over control, but the selective CaMKII inhibitor, KN-93, attenuated MKK3/6 and p38 as well as PKR and eIF2α phosphorylation. Although CaMKII was not affected by inhibition of PKR with 2-aminopurine, phosphorylation of MKK3/6 and p38 as well as eIF2α were significantly reduced. Collectively, these data provide evidence that CaMKII is a regulator of PKR-dependent p38 and eIF2α phosphorylation in response to ER calcium depletion by TZDs. Furthermore, using structural derivatives of TZDs that lack PPARγ ligand-binding activity as well as a PPARγ antagonist, we show that activation of these kinase signaling pathways is PPARγ-independent. The thiazolidinediones (TZDs) are synthetic peroxisome proliferator-activated receptor γ (PPARγ) ligands that promote increased insulin sensitivity in type II diabetic patients. In addition to their ability to improve glucose homeostasis, TZDs also exert anti-proliferative effects by a mechanism that is unclear. Our laboratory has shown that two TZDs, ciglitazone and troglitazone, rapidly induce calcium-dependent p38 mitogen-activated protein kinase (MAPK) phosphorylation in liver epithelial cells. Here, we further characterize the mechanism responsible for p38 MAPK activation by PPARγ ligands and correlate this with the induction of endoplasmic reticulum (ER) stress. Specifically, we show that TZDs rapidly activate the ER stress-responsive pancreatic eukaryotic initiation factor 2α (eIF2α) kinase or PKR (double-stranded RNA-activated protein kinase)-like endoplasmic reticulum kinase/pancreatic eIF2α kinase, and that activation of these kinases is correlated with subsequent eIF2α phosphorylation. Interestingly, PPARγ ligands not only activated calcium/calmodulin-dependent kinase II (CaMKII) 2-fold over control, but the selective CaMKII inhibitor, KN-93, attenuated MKK3/6 and p38 as well as PKR and eIF2α phosphorylation. Although CaMKII was not affected by inhibition of PKR with 2-aminopurine, phosphorylation of MKK3/6 and p38 as well as eIF2α were significantly reduced. Collectively, these data provide evidence that CaMKII is a regulator of PKR-dependent p38 and eIF2α phosphorylation in response to ER calcium depletion by TZDs. Furthermore, using structural derivatives of TZDs that lack PPARγ ligand-binding activity as well as a PPARγ antagonist, we show that activation of these kinase signaling pathways is PPARγ-independent. The thiazolidinedione (TZD) 1The abbreviations used are: TZD, thiazolidinedione; PPAR, peroxisome proliferator-activated receptor; ciglitazone, (+)-5-4-(1-methylcyclohexylmethoxy)benzylthiazolidine-2,4-dione; Δ2-ciglitazone, (+)-5-4-(1-methylcyclohexylmethoxy)benzylidenethiazolidine-2,4-dione; troglitazone, (+)-5-4-(6-hydroxy-2,5,7,8-tetramethylchroman-2-ylmethoxy)benzyl-2,4-thiazolidinedione; Δ2-troglitazone, (+)-5-4-(6-hydroxy-2,5,7,8-tetramethylchroman-2-ylmethoxy)benzylidene-2,4-thiazolidinedione; MAPK, mitogen-activated protein kinase; Erk, extracellular signal-regulated kinase; EGFR, epidermal growth factor receptor; CRNK, calcium-dependent tyrosine kinase-related non-kinase; ER, endoplasmic reticulum; PERK, PKR-like endoplasmic reticulum kinase/pancreatic eIF2α kinase; eIF2α, eukaryotic initiation factor 2α; PKC, protein kinase C; CaMKII, calcium/ calmodulin-dependent protein kinase II; PKR, double-stranded RNA-activated protein kinase; FBS, fetal bovine serum; Pyk2, proline-rich tyrosine kinase; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo3,4-dpyrimidine; PP3, 4-amino-7-phenylpyrazol3,4-dpyrimidine; TPA, 12-o-tetradecanoylphorbol-13-acetate; KN-93, 2-N-(2-hydroxyethyl)-N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine; KN-92, 2-N-(4-methoxybenzenesulfonyl)amino-N-(4-chlorocinnamyl)-N-methylbenzylamine phosphate; GW9662, 2-chloro-5-nitrobenzanilide; BAPTA, 1,2 bis(o-aminophenyoxy)ethane-N,N,N′,N′-tetraacetic acid; JNK, c-Jun NH - terminal kinase; Ad, adenovirus; PACT, PKR-activating protein. drug class was created over 20 years ago with the synthesis of ciglitazone, an analog of the hypolipidemic agent clofibrate (1Sohda T. Mizuno K. Imamiya E. Sugiyama Y. Fujita T. Kawamatsu Y. Chem. Pharm. Bull. 1982; 30: 3580-3600Crossref PubMed Scopus (199) Google Scholar). The TZDs have since grown to include three other members, troglitazone, rosiglitazone, and pioglitazone, which function to improve metabolic control in patients with type II diabetes by increasing insulin sensitivity in adipose tissue, muscle, and liver (2Olefsky J.M. J. Clin. Invest. 2000; 106: 467-472Crossref PubMed Scopus (508) Google Scholar). Following their discovery, it was learned that TZDs are ligands for the gamma isoform of the peroxisome proliferator-activated receptor (PPARγ) (3Lehmann J.M. Moore L.B. Smith-Oliver T.A. Wilkison W.O. Willson T.M. Kliewer S.A. J. Biol. Chem. 1995; 270: 12953-12956Abstract Full Text Full Text PDF PubMed Scopus (3459) Google Scholar). 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K. and Cancer in Interestingly, these derivatives growth inhibitory effects their Collectively, these data the of evidence that TZDs have or laboratory and have shown that TZDs mitogen-activated protein kinase (MAPK) activity J.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, T. R. M. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M. A. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The three extracellular signal-regulated kinase and c-Jun kinase are to in a of in R. PubMed Scopus Google Scholar). are activated a mechanism a MAPK kinase kinase or an MAPK kinase or which a p38 is by and J. T. J. 1995; PubMed Scopus Google Scholar, T. N. K. Y. K. T. K. Y. H. K. E. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google the other multiple are to a J.M. J. PubMed Scopus Google Scholar). This the and of these signaling pathways that MAPK activated both and in a In the is activated primarily by and p38 are activated by and of in phosphorylation of transcription that the expression of in addition to their PPARγ ligand-binding the ability of TZDs to induce MAPK phosphorylation an by which these agents cell growth and is to these agents induce MAPK signaling as well as to the of PPARγ in this the mechanism by which ciglitazone and activate of the MAPK in liver epithelial J.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Interestingly, data that PPARγ is not for TZD-induced MAPK phosphorylation. In the we have the structural derivatives of ciglitazone and that are of PPARγ ligand-binding activity in addition to a PPARγ to provide evidence that MAPK activation by TZDs in is PPARγ-independent. we have further the mechanism responsible for p38 activation by TZDs and provide evidence a p38 activation and induction of endoplasmic reticulum stress. thiazolidinediones ciglitazone and troglitazone, PP2, PP3, and were and Δ2-troglitazone, derivatives with attenuated PPARγ ligand-binding were as S. K. and Cancer in were as in and were to in with a of TPA, 2-aminopurine, and were was was and were MKK3/6 p38 PKR and eIF2α were was and were as J.W. J. 1998; PubMed Scopus Google Scholar, H. R. D. M. R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). was The the of Pyk2, T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google was and as A. P. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). liver epithelial were grown in with and as R. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). were and in with and as as well as and to were in the for the was and the were with The were and with and in PERK, PKR, and eIF2α were in with and The cell were by for was using the protein a of cell was in and for to were by and to were with the with and with for were with to the and by In were in for and with were as and The were by of cell was by with the of were to which were an were by and in was with a were in and by as In CaMKII kinase activity in was using a CaMKII to the with CaMKII was of cell in by with an CaMKII activity in was by the of the of to a CaMKII for was using and with a of and by we have shown that phosphorylation in liver epithelial J.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Interestingly, kinase activity was by a PPARγ that ciglitazone this signaling a derivatives of two and which lack PPARγ ligand-binding S. 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Chem. 1999; Full Text Full Text PDF PubMed Scopus Google we that Δ2-ciglitazone, ciglitazone, phosphorylation of with PP2, a selective kinase inhibitor, phosphorylation as well as In of with PP3, a analog of PP2, not the ability of to induce or phosphorylation. these data show that as well as phosphorylation by Collectively, these using a PPARγ provide evidence that TZDs activate kinase signaling pathways in cells. of the p38 MAPK addition to their effects the and Erk, we that both ciglitazone and rapidly activate p38 in J.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). PPARγ p38 phosphorylation in an and was correlated with in calcium and activation of the calcium-dependent tyrosine kinase Here, we and the ability to activate and Interestingly, both of the significantly in p38 with the of the kinase kinase of and phosphorylation and to activate these kinases as as ciglitazone and troglitazone, further the of PPARγ in p38 we with the PPARγ to with ciglitazone, troglitazone, or their to the ability of ciglitazone or to activate MKK3/6 and p38 of with and of the not the lack of MKK3/6 and p38 phosphorylation in response to these derivatives these that TZD-induced p38 is also PPARγ-independent. In of this TZDs and PPARγ and pioglitazone, were to activate p38 MAPK in not of as an of a and p38 MAPK, we further activation of was necessary for the effects of PPARγ ligands p38 phosphorylation. Specifically, were with a or a inhibitory of to with PPARγ an of T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google which to R. J. Biol. 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PPARγ MKK3/6 and p38 are by of a that activation in response to these these that an kinase is by PPARγ to p38 phosphorylation. to Pyk2, calcium/calmodulin-dependent kinase II (CaMKII) and the protein kinase and other kinases that activated by PPARγ the of in p38 activation by PPARγ were by of to Although depletion activation not ciglitazone and p38 phosphorylation was not affected This that are not involved in PPARγ p38 CaMKII is necessary for p38 phosphorylation in response to PPARγ the ability of ciglitazone and to activate p38 in the of the selective CaMKII inhibitor, K. K. T. A. M. T. H. Biochem. Biophys. Res. Commun. 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PubMed Scopus Google significantly PPARγ CaMKII In to effects CaMKII, and to TZD-induced phosphorylation Collectively, these that CaMKII, and not Pyk2, of MKK3/6 and p38 in this signaling of CaMKII by PPARγ ligands and their ciglitazone or as ciglitazone or as ciglitazone or as ciglitazone or as ciglitazone or as ciglitazone or as ciglitazone or as ciglitazone or as ciglitazone or as in a and were effective MKK3/6 and p38 their TZDs in MKK3/6 and p38 phosphorylation in response to ciglitazone and was we the to activate CaMKII kinase that and were significantly CaMKII their data further that CaMKII is necessary for p38 activation in response to PPARγ ligands and is consistent with the of to induce MKK3/6 and p38 phosphorylation. PPARγ ER and eIF2α in and were to rapidly calcium by calcium the endoplasmic reticulum (ER) H. R.M. Cancer Res. Google Scholar). is involved in protein and the ER that depletion to the of and subsequent ER N. J. Biol. Chem. 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TZDs with PPARγ not activate p38 in this cell these that activation of the p38 MAPK in by ciglitazone and is to a for we that the of the to activate p38 an of these to calcium the This is in the mechanism of PPARγ p38 we that p38 and are activated by but pathways Although the of as an of p38 MAPK has been in response to J.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A. P. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, P. S. S. A. A. A. H. S. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google the lack of a selective has this to inhibitory of is a to with the protein for necessary for activation R. J. Biol. 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Full Text Full Text PDF PubMed Scopus Google and phosphorylation in response to these not this further evidence that PPARγ ligands activate two kinase pathways in cells. that the calcium for activation of these kinases in is the endoplasmic reticulum and were shown to promote calcium the ER to PKR-dependent phosphorylation of eIF2α, and growth H. R.M. Cancer Res. Google Scholar). these that PPARγ ligands ER stress. In of this we show that TZDs not only induce eIF2α phosphorylation but also activate and Although PKR activation was H. R.M. Cancer Res. Google this is the evidence that these also is phosphorylation was only in response to ER and not H.P. Zhang Y. D. 1999; PubMed Scopus Google Scholar). and PKR are for ER eIF2α phosphorylation J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, H.P. Zhang Y. A. H. D. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). a attenuated PPARγ eIF2α phosphorylation in cells. Collectively, these data that calcium by PPARγ ligands in with protein in the ER ER stress. have to that in ER calcium are not only for ER but also provide a the of calcium for PPARγ CaMKII, Pyk2, and p38 activation for p38 as a of ER has been this MAPK was shown to multiple ER transcription transcription factor transcription factor and growth and to in activity D. PubMed Scopus Google Scholar, S. Biochem. J. PubMed Google Scholar, J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). In we provide evidence that ER stress-responsive eIF2α kinases are not only activated by PPARγ but that PKR to PPARγ MKK3/6 and p38 Although PKR has been as an of p38 in the response J. 2000; PubMed Scopus Google this is the evidence that PKR ER p38 phosphorylation. Although the mechanism by which PKR p38 it was shown that PKR with in response to double-stranded a and p38 phosphorylation M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). with the data using that PKR as an of p38 However, not the of kinases that also to p38 phosphorylation in this Specifically, PKR activation in response to PPARγ but MKK3/6 and p38 phosphorylation is Furthermore, the for PKR activation in that of MKK3/6 and Although this to a lack of sensitivity in the in the are necessary to a PKR and p38 as in cell these data the ER as a in TZD-induced signaling in are to a for the ER in this Interestingly, the data a for both CaMKII and PKR as of the p38 MAPK and eIF2α pathways in response to PPARγ Although double-stranded is the PKR the activity of this kinase is also increased in double-stranded by a protein PKR-activating protein as J. 1998; PubMed Scopus Google Scholar, T. M. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). In response to as is and with PKR to in kinase activity and eIF2α phosphorylation T. M. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Although phosphorylation of CaMKII the kinase responsible for phosphorylation has not been we that CaMKII PKR in response to PPARγ PKR MKK3/6 and p38 as well as eIF2α phosphorylation with their to activate CaMKII, Pyk2, and also to induce phosphorylation to the as their in the pathways we in and ciglitazone and only in their ability to activate the Although we have shown that PPARγ and p38 phosphorylation in is calcium-dependent J.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google activation of PERK, PKR, and CaMKII in response to calcium has been in the the other KN-93, and to significantly PPARγ and phosphorylation not This observation that activation of this signaling is and Δ2-ciglitazone, which does not activate and phosphorylation to the as that the of the to induce phosphorylation of kinases in is to their to promote ER calcium with this and were to induce eIF2α phosphorylation to the as the In we provide evidence that the PPARγ ligands ciglitazone and activate CaMKII and that this kinase, as to Pyk2, is for MKK3/6 and p38 phosphorylation. of is correlated with phosphorylation of the ER kinase and subsequent eIF2α phosphorylation. CaMKII as a regulator of PKR in response to ER and that PKR a necessary in activation of the p38 in response to ER calcium The ability of ciglitazone and to induce ER as well as activation of an in their effects cell growth and with we that both ciglitazone and significantly cell not In and not only to induce and eIF2α phosphorylation in this cell but also were to significantly cell that these kinases a necessary in PPARγ cell Collectively, these that TZDs induce signaling that have to the mechanism responsible for their activity in a of