Modulation of Activity

Regulation by Phosphorylation

1. downregulation by phosphorylation

a) Ser112 phosphoryaltion by ERK (MAPK) (Hu, Kim et al. 1996; Zhang, Berger et al. 1996; Adams, Reginato et al. 1997; Camp and Tafuri 1997)

1) EGF induces phosphorylation of PPARg by ERKs (Zhang, Berger et al. 1996)

2) TPA induces phosphorylation of PPARg by ERKs (Zhang, Berger et al. 1996)

3) TNFa can activate ERK2 and JNK to cause phosphorylation (Adams, Reginato et al. 1997)

4) PPARg2 Ser112Asp properties (Shao, Rangwala et al. 1998)

a) decreased ligand-binding activity and coactivator recruitment

b) may play an important role in the conformation of the unliganded receptor, explaining the change in regulation perhaps

c) may be responsible for altering the ability of PPARg to respond to ligands, or to bind ligands

5) phosphorylated to regulate activity

a) phosphorylated in vitro by addition of Erk-1 (Hu, Kim et al. 1996)

b) phosphorylation induced by insulin, EGF, TPA, and serum (Hu, Kim et al. 1996)

c) TPA induced phosphorylation to inhibit the induction of PPARg by pioglitizone (Hu, Kim et al. 1996)

d) delays differentiotion by a few days in cultured NIH3T3 cells (Hu, Kim et al. 1996)

b) Ser82 phosphorylated by c-JNK to negatively regulate activity(Camp, Tafuri et al. 1999)

1) corresponds to Serine 112 on mPPARg2

2) phosphorylated in response to PDGF to negatively regulate PPARg function

3) mutagenesis of this site prevents the negative regulation (Hu, Kim et al. 1996; Camp and Tafuri 1997)

c) Ser110 phosphorylation on PPARg (Hu, Kim et al. 1996)

1) MAPK phosphorylates PPARg, leading to a decrease in its transcriptional activity at Ser 110 in adipocytes

2) MAPK is stimulated by growth factors that inhibit fat cell differentiation

3) mutagenesis at Ser112 to stop this phosphorylation prevents negative regulation of differentiation

d) Ser84 phosphorylation on PPARg (Adams, Reginato et al. 1997)

1) phosphorylated in vivo, but stopped by mutagenesis to alanine

2) ERK2 and JNK can phosphorylated Ser84 in vitro

3) Gal4-PPARg A/B chimera is more transcriptionally active if the serine is converted to alanine at Ser84

4) S84A is more able to induce adipogenesis

5) role: phosphorylation through a MAPK dependent pathway seems to repress the activity PPARg

e) PPARg1 is phosphorylated in vivo in response to PDGF and EGF treatment (Camp and Tafuri 1997)

1) PPARg1 is phosphorylated in vitro by recombinant MAPK

2) single potential MAPK site at Ser82

3) phosphorylation of this site reduces transcriptional activity of PPARg1 in response to growth factor treatment

f) PPARg is phosphorylated in vivo (Zhang, Berger et al. 1996)

1) aP2 gene is stimulated in rat adipocytes and 3T3-L1 cells by TZD and insulin

a) cotransfection with dominant-negative MKK1 stops the stimulation of this gene

b) constransfection with constitutively active NKK1 activates the stimulation of this gene

3) insulin and constitutively active MKK1 both induces phosphorylation of PPARg

g) PGF2a inhibits PPARg activity through a MAPK dependent manner (Reginato, Krakow et al. 1998)

1) 3T3-L1 cells, PGF2a prevents adipogenesis normally

2) 30 minute treatment in 3T3-L1 cells causes induction and phosphorylation of MAPK (Reginato, Krakow et al. 1998)

3) PGF2a prevents the differention of 3T3-L1 cells, and this can be stopped with treatment of PD98059

4) mutated S112A PPARg2 is immune to the inhibitory effects of PGF2a through MAPK, and is still able to induce adipogenesis (Reginato, Krakow et al. 1998)

h) 15-LO-1 (Hsi, Wilson et al. 2001)

1) overexpression of 15-LO-1 in HCT-116 cells down-regulates PPARg through the MAPK signaling pathway

a) 13-HODE, produced by 15-LO-1, upregulates MAPK activity and negative PPARg phosphorylation (Hsi, Wilson et al. 2001)

i) Pro12ala polymorphism

1) associated with reduced risk of type 2 diabetes and insulin resistance

2) reduced risk of diabetes and insulin resistance in twins

a) significant impact of the pro12ala on glucose tolerance, diabetic status, homeostasis model assessment for insulin resitance and plasma insulin profile in twins (Poulsen, Andersen et al. 2003)

j) 13-(S)-HODE increased PPARg phosphorylation (Hsi, Wilson et al. 2002)

k) 15-(S)-HETE decreases PPARg phosphorylation (Hsi, Wilson et al. 2002)

2. phorbol esters

a) TPA induces expression of PPARg in primary macrophages and THP-1 monocytic leukemia cells (Ricote, Huang et al. 1999)-25

1) inhibited by blocking PKC

b)

Protein Modifications

1. nitration

a) peroxynitrate treatment of RAW 264 cells (ONOO-, 500mM, 40min) causes nitration (Shibuya, Wada et al. 2002)

b) prevents ligand-induced translocation to the nucleus (Shibuya, Wada et al. 2002)

2. ubiquitination

a) PPARg is ubiquitinated to cause its degradation (Hauser, Adelmant et al. 2000)

PPARg Interaction with Coactivators and Proteins

1. NCoR

a) role

1) corepressor; mediates the effects of insulin on inhibition of PPARg

a) anti-NCoR blocks trans-repression of PPARg by insulin (Lavinsky, Jepsen et al. 1998)

b) interaction site

1) hinge region (Zamir, Zhang et al. 1997)

c) interaction

1) weak interaction with NCoR and SMAR (Wang, Fu et al. 2001)-26

2. SRC-1

a) role

1) enhances PPAR:RXR transactivation

a) overexpression of a mutant SRC-1 which has only the nuclear receptor-interacting domain represses ligand-dependent transcription by PPAR:RXR (Zhu, Qi et al. 1996; DiRenzo, Soderstrom et al. 1997)

b) anti SRC-1 antibodies microinjected stops TZD-dependent activation of PPRE reporter in Rat-1 cells (Westin, Korokawa et al. 1998)

1) stopped by the addition of extra SRC-1 (Westin, Korokawa et al. 1998)

2) bind and enhances transactivation by PPAR in a ligand-dependent manner (Zhu, Qi et al. 1996)

a) interacts with PPARg through a central region which contains three conserved helical motifs of the consensus LXXLL (Kamei, Xu et al. 1996; Yao, Ku et al. 1996; Torchia, Rose et al. 1997; Kalkhoven, Valentine et al. 1998; Voegel, Heine et al. 1998)

b) second LXXLL motif of SRC-1 seems to dictate which nuclear receptors are preferred

c) K301A (helix 3), V315A (helix 4), Y320A (helix 5), and L468A and E471A (helix 12) lose their ligand-induced coactivator interaction function (Chen, Johnson et al. 2000)-17

b) interaction site

1) interact via two different domains on SRC-1 (Zhu, Qi et al. 1996)

a) liganded PPARg LBD has been crystallized with a region of SRC-1 (amino acid 623-710) encompassing the two LXXLL motifs (Nolte, Wisely et al. 1998)

b) rosiglitazone induces the two LXXLL motifs of a isngle SRC1 molecule to interact separately with the AF2 of each receptor, making a stable ternary complex: two PPARg LBDs and 1 SRC1 molecule (Nolte, Wisely et al. 1998)

1) LXXLL helix is orieted by a conserved glutamic acid of the AF2 helix and a conserved lysine in helix3 (K301) of the LBD, placing the LXXLL motif in the hydrophobic pocked formed by helices 3, 4, 5, and AF2 of PPARg (Hsu, Palmer et al. 1995)

2) distinct amino acids C-terminal to the core LXXLL motif are required for PPARg activation in response to different ligands (McInerney, Rose et al. 1998)

2) PPAR requires E471 to bind with SRC-1

a) mutation of that site to E471A stops transactivation and interaction of PPAR with SRC-1 (Zhou, Cummings et al. 1998)

3) SRC-1 interacts with PPARg (DiRenzo, Soderstrom et al. 1997; Krey, Braissant et al. 1997; Li, Gomes et al. 1997; Zhu, Qi et al. 1997)

3. p300

a) role

b) PPARg interaction

1) interacts through LXXLL motifs (Wang, Fu et al. 2001)-56

a) contacts AF-2 in a ligand-dependent manner (Wang, Fu et al. 2001)-24

b) contacts AF-1 in a ligand-independent manner (Wang, Fu et al. 2001)-24

 

2) domain interactions

a) p300 interacts with the DEF domain in a ligand-dependent manner

b) p300 interacts with the ABC domain in a ligand-independent manner

1) amino acids 31-99 (Gelman, Zhou et al. 1999)

c) p300 can coactivate both the AF-1 and AF-2 domains (Gelman, Zhou et al. 1999)

4. CBP

a) role

b) interaction site

1) interaction with CBP and SRC-1 requires K301A, V315A, Y320A, L468A, and E471A (Chen, Johnson et al. 2000)

c) CBP associates in response to ligand treatment

1) enhanced in a pulldown assay by the presence of BRL (Li, Pascual et al. 2000)

a) terminal 450 amino acids of CBP are required for activation of the AOX reporter (Li, Pascual et al. 2000)

d) requires other coactivators to bind to PPARg

1) requires SRC-1 to bind well to PPARg (Li, Pascual et al. 2000)

2) BRL49653 promotes PPARg/RXRa/CBP interactions (Schulman, Shao et al. 1998)

e) coactivates PPARg

1) NIH3T3 cells transfected with PPARg and increasing CBP had increased response to TZD in reporter assays (Takahashi, Kawada et al. 2002)

a) endogenous aP2 and LPL were also activated in the same manner (Takahashi, Kawada et al. 2002)

b) ribozyme targeting of CBP likewise inhibited PPARg-mediated induction of aP2 and LPL (Takahashi, Kawada et al. 2002)

c) ribozyme targeting of CBP also inhibits adipocyte differention from these cells (Takahashi, Kawada et al. 2002)

5. Is coreceptor interaction required for PPARg transactivation?

c) K301A (helix 3), V315A (helix 4), Y320A (helix 5), and L468A and E471A (helix 12) lose their ligand-induced coactivator interaction function (Chen, Johnson et al. 2000), but can still bind ligand

6. ligand-dependent specific coactivator recruitment to PPARg

a) PGJ2 induces interactions with SRC-1, TIF2, AIB-1, p300, TRAP220/DRIP205 in yeast and M2H assays (Kodera, Takeyama et al. 2000)

b) troglitazone does not induce interactions of PPARg with coactivators (Kodera, Takeyama et al. 2000)

7. TRAPcomplex

a) TRAP220 subunit of the TRAP complex interacts with PPARg2 (Ge, Guermah et al. 2002)-3,4

b) TRAP 220 interaction with PPARg2 is required for adipocyte differentiation (Ge, Guermah et al. 2002)

1) mouse embryonic fibroblasts cannot differentiate to adipocytes in the absence of PPARg2 (Ge, Guermah et al. 2002)

 

8. FABP

b) regulation of PPARg

1) transfection of COS-7 cells with adipocyte-FABP (A-FABP) causes a greater induction of a reporter in vitro than with just ligand alone (Tan, Shaw et al. 2002)

a) dose-dependent, but plateaus at 0.3ug of FABP

2) L-FABP interacts with PPARa and PPARg, and down-regualtion of FABP lowers the transcriptional activity of all three PPAR subtypes (Tan, Shaw et al. 2002)-49

3) A-FABP selectively enhances the activity of PPARg (Tan, Shaw et al. 2002)

a) FABPs massively relocate to the nucleus in response to selective ligands for PPARg (Tan, Shaw et al. 2002)

b) directly interact with PPARg in a receptor- and ligand-selective manner (Tan, Shaw et al. 2002)

9. PBP

a) regulation of PPARg

1) overexpression slightly enhances PPARg transactivation (Yuan, Ito et al. 1998)

2) overexpression of truncated form has a dominant negative effect on PPARg transactivation (Yuan, Ito et al. 1998)

 

 

Ligand Independent Activation

1. DNA interactions

a) even without ligand, PPARg/RXRb can bind to DNA (Kodera, Takeyama et al. 2000)

1) TIF2 binds in the presence of ligand, creating a larger complex (Kodera, Takeyama et al. 2000)

Modulation by Physiological Changes

1. insulin

** see Insulin section below for more information on insulins effects

a) alters transactivation by PPARg (Hu, Kim et al. 1996; Zhang, Berger et al. 1996; Werman, Hollenberg et al. 1997)

1) CV-1 cells endogenous PPARg activation by BRL is not enhanced by insulin, but transfected PPARg2 shows a large increase in transactivation of a PPARg reporter (Shalev, Siegrist-Kaiser et al. 1996)

b) alters PPARg1/2 levels

1) PPARg1/2 are decreased in rodents by fasting and insulin deficiencies (Cusin, Terrettaz et al. 1990; Appel and Fried 1992; Girard, Perdereau et al. 1994; O'Brien and Granner 1996; Vidal, Auboeuf et al. 1996)

2) insulin induces PPARg mRNA (Chevalier and Roberts 1998)-187

1) PPARg expression is induced by insulin (Kersten 2001)-(Vidal-Puig 1997)

3) PPARg1 mRNA levels in abdominal subcutaneous adipose tissue does not correlate with BMI or fasting insulinemia (Chevalier and Roberts 1998)-171,195

4) insulin activates PPARg2 after 2 hours, but returns to basal after three hours (Rieusset, Andreelili et al. 1999)

5) PPARg1 slowly increases after insulin treatment (Rieusset, Andreelili et al. 1999)

c) induces phosphorylation of PPARg

1) ERKs: transfection with a dominant negative MEK decreased the effects of both insulin and TZD on PPARg activity (Zhang, Berger et al. 1996)

2. cold acclitimization

a) PPARg mRNA expression is lowered during cold acclimatization in rats as measured by Northern blots (Guardiola-Diaz, Rehnmark et al. 1999) during the first two weeks, but return to near-normal levels during the following two weeks

3. diet

a) high fat diet

1) PPARg is induced in rats fed high fat diets by oral gavage for at least four days, but not in animals fed high carbohydrate diets (Chevalier and Roberts 1998)-191

2) in mice, high fat diet increases PPARg expression in adipose tissue of normal mice, and induces PPARg2 expression in the livers of obese mice (Chevalier and Roberts 1998)-193

b) fasting

1) for 48 hours reduces the expression of PPARg isoforms in subcutaneous and visceral adipose tissue (Chevalier and Roberts 1998)-192

2) in mice, fasting and insulin-deficient diabetes both reduce the level of PPARg expression (Chevalier and Roberts 1998)-193

17.3 diet and humans

3) PPARg1 mRNA levels in abdominal subcutaneous adipose tissue does not correlate with BMI or fasting insulinemia (Chevalier and Roberts 1998)-171,195

4) comprehensive report of PPARg expression changes in response to starvation (Escher, Braissant et al. 2001)

a) no changes, except for decrease in ileum (Escher, Braissant et al. 2001)

b) fasting for 7.5 and 12 hours inhibits expression of PPARb mRNA by 50% in liver of rats (Escher, Braissant et al. 2001)

c) fasting for 7.5 and 12 hours induced in time-dependent manner the expression of PPARb in the kidney (Escher, Braissant et al. 2001)

c) obesity

1) causes the adipose tissue of humans to have increased expresson of PPARg2 mRNA, an increased ratio of PPARg2/g1 ratio in proportion to BMI (Chevalier and Roberts 1998)-187

2) increased PPARg2/g1 is also seen in obese rhesus monkeys (Chevalier and Roberts 1998)-194

d) low calorie diet

1) down-regulates the expression of PPARg2 mRNA in adipose tissue of obese humans (Chevalier and Roberts 1998)-194

4. HDL

a) role

b) regulation of PPARg

1) HDL inhibits mRNA expression of CD36 and aP2 in RAW cells in vitro (Han, Hajjar et al. 2002)

2) PPARg expression is enhanced in fibroblasts and RAW cells by exposure to HDL for 10 hours (Han, Hajjar et al. 2002)

3) PPARg upregulates transcription in response to HDL treatment (Han, Hajjar et al. 2002)

a) increased migration to nucleus after HDL exposure (50ug/ml) for 16 hours

b) treatment with U1026 (MAPK inhibitor) blocks this inhibition

4) HDL treatment blocks PPARg stimulated adipocyte differentiation

a) MAPK inhibitor stops this

Modulation by Other NHR Pathways

1. GR

a) corticoisteroids (GR) induce PPARg mRNA (Chevalier and Roberts 1998)-187

2. RXRa

a) RXRa ligands

1) ligation of RXRa ligands also can promote transcription in a PPAR-dependent manner

a) 9-cis-RA and other RXR ligands display antidiabetic activity similar to that of TZD (Shao, Rangwala et al. 1998)

2) RXRa ligand LG100268 (100nM) treatment of 3T3L-1 preadipocytes induces lipid accumulation (oil red O) similarly to BRL49653-mediated (100nM)

3) cotreatment with LG100268 and BRL49653 causes a synergistic effect

a) accumulation of lipids in 3T3-L1 preadipocytes (Schulman, Shao et al. 1998)

b) activation of PPREx3-TK-Luc in NIH 3T3 cells (Schulman, Shao et al. 1998)

3. ER

a) duck uropygial gland (Ma, Sprecher et al. 1998)

1) expresses high amounts of PPARg1

2) estradiol treatment to mallard ducks causes peroxisome proliferation in this tissue (Ma, Sprecher et al. 1998)-4

3) PGD2 levels decrease, and d12-prostaglandin J2 (PGJ2) levels increase

4. retinoic acid

a) in brown fat, treatment with 9-cis-RA or all trans RA decreased the expression of PPARg mRNA (Valmaseda, Carmona et al. 1999)

5. PPARa ligands

a) wy-14,643

1) treatment of brown adipocytes with 10mM Wy for 24 hours decreases the amount of PPARg mRNA

2) treatment of activated T cells with Wy-14,643 (250mM) for 72 hours induces PPARg expression (Cunard, Ricote et al. 2002)

 

Regulation of PPARg Expression

1. PPARg autoregulates (Pearson, Cawthorne et al. 1996)

2. gene regulation of PPARg

a) C/EBP site is found in human PPARg2 promoter (Saladin, Fajas et al. 1999)

3. ADD1/SREBP-1c

a) role in adipogenesis (A. Transcription Factors)

a) co-transfection with ADD-1 further stimulates the reporter activity

1) aP2 reporter, PPARg, RXRa expression vectors,

2) NIH 3T3 cell transfection model (Kim, Wright et al. 1998)

b) dominant negative ADD1 can be countered by the addition of PPARg ligands (Kim, Wright et al. 1998)

c) ADD1 causes the release of a soluble factor that activates PPARg (Kim, Wright et al. 1998)

1) factor can compete away the binding of BRL 49653

d) ADD1/SREBP directly regulates the transcription of PPARg (Fajas, Schoonjans et al. 1999)

1) SREBP may produce an endogenous ligand for PPARg (Kim and Spiegelman 1996; Kim, Wright et al. 1998)

e) overexpression of nuclear SREBP1c in adipose tissue of transgenic mice lowers PPARg expression and other markers of adipocyte differentiation (Desvergne and Wahli 1999)-273

f) SREBP2

1) has a binding site in the PPARg3 promoter, and cholesterol depletion stimulates this promoter (Fajas, Schoonjans et al. 1999)

4. artificial zinc finger proteins

a) ZFP54

1) PPARg2 and PPARg1 transcription is inhibited by ZFP54 when transfected into 3T3-L1 adipocytes (Ren, Collingwood et al. 2002)

a) completely stops differentiation of 3T3-L1s to adipocytes

b) ZFP55

1) only PPARg2 is inhibited by ZFP55 when transfected into 3T3-L1 preadipocytes (Ren, Collingwood et al. 2002)

a) stops most differentiation of 3T3-L1s into adipocytes

5. C/EBPa

a) can induce PPARg2 expression by direct binding to specific sites in the PPARg promoter (Rosen, Hsu et al. 2002)-(Elberg 2000)

6. Egr-1

a) role

b) regulation of PPARg

1) overexpression of Egr-1 activates the human PPARg promoter in VSMC and HepG2 cells (Fu, Zhang et al. 2002)

2) Egr-1 binds an Egr-1 element in the PPARg promoter prior to activation (Fu, Zhang et al. 2002)

Modulation by Inflammatory Molecules

1. TNFa

a) TNFa inhibits adipocyte differntiation and antagonizes PPARg gene expression (Zhang, Berger et al. 1996)

b) TNFa triggers dedifferentiation of adipocytes and reduces the expression of adipocyte specific genes (Chevalier and Roberts 1998)-188-189

c) TZD, insulin sensitizer, opposes TNFa-mediated repression of adipocyte genes (Clarke, Robinson et al. 1997)

2. IL-4

a) induces PPARg1 expression in monocytes and macrophages (Chinetti, Fruchart et al. 2000)-30

b) activated T cells treated with IL-4 have a modest increase in PPARg after 72 hours of treatment (Cunard, Ricote et al. 2002)

3. oxidized LDL products

a) 9- and 13- HODE increase PPARg1 mRNA levels (Chinetti, Fruchart et al. 2000)-25

1) 9-HODE and 13-HODE are both ligands as well (Chinetti, Fruchart et al. 2000)-37

b) induced in primary macrophages and monocytic cell lines by ox-LDL (Ricote, Huang et al. 1999)-25

1) no effect of PKC inhibitors on this induction (Ricote, Huang et al. 1999)-25

c) oxLDL and lipid peroxisdation derivatives up-regulate and activate PPARg in monocytes (Nagy, Tontonoz et al. 1998; Tontonoz, Nagy et al. 1998)

1) autoregulatory, since PPARg activates CD36 and SRA gene transcription, allowing in more oxLDL to activate PPARg

4. cytokines

a) oxLDL, M-CSF, and GM-CSF induce expression of PPARg in monocytic cell lines and primary macrophages (Ricote, Huang et al. 1999)-25

Regulation by Growth Factors

1. PDGF

a) treatment of adipocytes in culture decreases the transcriptional activity of PPARg1 (Camp and Tafuri 1997)

Regulation by Transcription Factors

1. C/EBP

a) C/EBPb expression with dexamethasone treatment induces PPARg expression

1) presumably through C/EBP sites within the promoter region of PPARg2 gene (Clarke, Robinson et al. 1997)

b) mice null for both C/EBPb and C/EBPd, normal C/EBPa, have normal PPARg expression, but impaired adipogenesis (Tanaka, Yoshida et al. 1997)

c) overexpression of a dominant negative protein for all three C/EBPs (A-ZIP/F-1) allows normal PPARg expression but no fat development (Desvergne and Wahli 1999)-269

d) adipocyte conversion of 3T3 fibroblasts require C/EBPb, C/EBPd, and dexamethasone to induce PPARg expression (Wu, Bucher et al. 1996)

2. SREBP-1

a) PPARg expression is induced by SREBP-1 (Fajas, Schoonjans et al. 1999)

Mutation Effects on PPARg Function

see Structure section

Other modulation of activity means

1. protein degradation

a) activation of PPARg by its ligands causes proteolytic degradation of the receptor itself (Panigrahy, Singer et al. 2002)-27

1) also true in endothelial cells

2) cotreatment with lactacystin, a proteosome inhibitor, stops the ligand-dependent degradation of PPARg (Panigrahy, Singer et al. 2002)

2. PLA2

a) role causes release of arachidonic acid

b) upregulates PPARg activity in epithelial lung cells

1) overexpression causes an increase in PPARg reporter activity (Pawliczak, Han et al. 2002)

2) PLA2 inhibitor arachidonyltrifluoromethyl ketone lowers PPARg activity, ut other cPLA2 inhibitors like LY311727 had no effect (Pawliczak, Han et al. 2002)

3) calcium ionophore A23187 also increased PPARg activity (Pawliczak, Han et al. 2002)