VI.  RXRa = BD73

A.  Isoforms

            1.  there are three subtypes

                        RXRa, RXRb, and RXRg (reviewed in (Canan Koch, Dardashti et al. 1996)-5)

            2.  RXRb info

                        a)  ligands

                                    1)  phytanic acid and docosahexaenoic acid, and lithocholic acid compete with 9-cis-RA for binding on RXRb

B.  Heterodimerization

            1.  ligand binding to RXRa preferentially promotes the formation of RXR homodimers (Desvergne and Wahli 1999)-114

            2.  heterodimerization with other nuclear receptors (Schulman, Li et al. 1997)-(Leblanc 1995)

                        a)  permissive with some and nonpermissive with others

                                    1)  permissive - PPAR, LXR, and NGFIB

                                    2)  nonpermissive – RAR, TR

                                                a)  dimerization with these partners causes a decrease in the ability of RXR to bind hormone (Schulman, Li et al. 1997)-(Kurokawa 1994, Forman 1995)

                                                b)  also prevents RXR from binding with its ligand (Schulman, Li et al. 1997)-(Kurokawa 1994, Forman 1995)

                        b)  AF-2 domain of RXR is important for the transactivation of partners

                                    1)  deletion of the RXR AF-2 domain reduces activation of TR by its hormone (Schulman, Li et al. 1997)-(Leng 1995, Schulman 1996)

                                    2)  can recruit corepressors (Schulman, Li et al. 1997)-(Schulman 1996)

                        c)  RXR is the heterodimeric partner for many Class II hormone receptors


            3.  RXRa is the heterodimerization partner for PPARs (Heyman, Mangelsdorf et al. 1992; Levin, Sturzenbecker et al. 1992)

            4.  PPAR heterodimerizes with RXR

                        b)  discovery – PPAR acts synergistically with RXR (Kliewer, Umesono et al. 1992) and drives a reporter upstream of a heterologous acyl-CoA oxidase (ACO)PPRE

                                    1)  later proven in a natural setting (Issemann, Prince et al. 1993; Keller, Dreyer et al. 1993)

                        a)  PPAR binds with the retinoid X receptor (RXR) (Kliewer, Umesono et al. 1992; Gearing, Gottlicher et al. 1993; Mangelsdorf and Evans 1995)


C.  Role

C1.  Expression

            1  expression pattern

                        a)  Rev-erba is induced only in the liver, not intestine (Staels, van Tol et al. 1992)

                        b)  RevErba mRNA levels are dramatically increased during differentiation of preadipocytes to adipocytes (Chawla and Lazar 1993)

            2.  RXRa, b, and g have variable tissue distribution (Clark 2002)-11,12


            1.  differentiation

                        a)  fat cell differentiation

                                    1)   RXRa, though not measureable in pre-adipocytes (undifferentiated 3T3-L1 cells), is induced several fold upon fat cell differentiation (Thuillier, Baillie et al. 1998) (Chawla and Lazar 1993)

                        b)  muscle cell differentiation

                                    1)  expression in myoblasts induces muscle differentiation – (Downes, Carozzi et al. 1995)

                        2.  ligands

                        a)  9-cis retinoic acid binds to RARs (Mangelsdorf, Ong et al. 1990; Heyman, Mangelsdorf et al. 1992; Levin, Sturzenbecker et al. 1992)

            2.  negative regulation of other nuclear receptors

                        a)  Rev-erba competes with TR for binding to TREs (Downes, Carozzi et al. 1995)

                                    1)  Rev-erba binds to TREs consisting of DR-4 sequences (Spanjaard, Nguyen et al. 1994)

C3.  Apoptosis

            1.  anti-apoptosis

                        a)  prevents H2O2 induced apoptosis

                                    1)  treatment with all-trans retinoic acid prevented prevented H2O2 induced apoptosis (Konta, Xu et al. 2001)

                                                a)  inhibited by treatment with HX531 (2.5mM), an antagonist of RXRa (Konta, Xu et al. 2001)

C4.  Retinoids Role

            1.  retinoids and their role

                        a)  inhibit the growth of cancer cells (Stio, Celli et al. 2001)-5

                        b)  with vitamin D analogues, induce additive growth inhibition of myeloma cells, breast cancer cells (Stio, Celli et al. 2001)-6, and LNCaP prostate cells (Stio, Celli et al. 2001)-7

                        c)  causes hypercalcemia, hypercalciuria, and soft tissue calcification

                                    1)  work to develop analogues that have changes in the side chain that lower the calcemic activity

                                                a)  may be more effective that 1,25(OH)2D3 at modulating the proliferation and differentiation of various malignant and non-malignant cells (Stio, Celli et al. 2001)-8-13

                                                b)  EB 1089 and KH 1060 both have potent anticancer or immunosuppressive properties (Stio, Celli et al. 2001)-14,15

D.  Targets

            1.  AP-1

                        a)  AP-1 is induced in cells treated with H2O2, and this induction is repressed by treatment with all-trans-RA

                        b)  cotreatment with HX531, an RXR-specific inhibitor, prevents the effect on fos but not jun (Konta, Xu et al. 2001)

            2.  myc

                        a treatment of SH-SY5Y human neuroblastoma cells with 0.1 and 1 mM 9-cis RA decreases the level of c-myc protein dramatically (Stio, 2001, 213-222)

                                    1  cotreatment with vitamin D analogues synergistically inhibit myc protein levels (Stio, 2001, 213-222)

            3.  RXRa induces expression of RAR in response to RA treatment

                        a)  anisomycin, or a constitutive active mutant of MKK4 (SEK1) stops the RA induced induction of RARb (Kyriakis 2000)-16

                        b)  MKK4 directly phosphorylated RBRa in a COOH-terminal region (Kyriakis 2000)-16

            4.  apoC-3

                        a)  RXRa ligands activate this gene (Vu-Dac, Gervois et al. 1998)

            5.  CRBPII

                        a)  contains an RXRRE and transcription is enhanced by binding of an RXRa homodimers (Tanaka, Hora et al. 2003)-28

                        b)  expression is enhanced by treatment of mice with clofibrate, presumably because PPARa stabilizes RXRa protein expression in vivo (Tanaka, Hora et al. 2003)

E.  Modulation of Acitivity

E1.  Phosphorylation

            1.  ERK-catalyzed phosphorysation of RXRa prevents the stimulus-mediated induction of the VDR receptor (Kyriakis 2000)-12

                        a)  occurs through phosphorylation at Ser260 (Kyriakis 2000)-12

                                    1)  unknown whether this is due to ubiquitinylation and degradation

E2.  Coactivator

            1.  SRC-1

                        a)  RXRa prefers SRC-1 recruitment to CBP in a mammalian two-hybrid approach using LG100268 (Schulman, Shao et al. 1998)

            2.  coactivator and corepressor interactions

                        a)  coactivator pocket of RXR is essential to coactivator binding and transactivation by the RXR homodimer (Leo, Yang et al. 2001)

                                    1) NOT sufficient for transactivation of the RAR/RXR heterdimer (Leo, Yang et al. 2001)

                                    2)  deletion of this domain enhances the coactivator RAC3 recruitment of the RAR/RXR heterodimer (Leo, Yang et al. 2001)

                                    3)  deletion of the AF-2 helix of RAR abolishes RAC3 binding and transcriptional activation (Leo, Yang et al. 2001)

            3.  PGC-1

                        a)  PGC coactivates all RXRs in HeLa cells transfected with DR1 construct in a dose-dependent and ligand-dependent manner (Delerive, Wu et al. 2002)


E3.  Physiological Stimuli

            1.  hypoxia

                        a)  hypoxic conditions reduce the expression of RXRa

                                    1)  prevent activation of an RXRE in cardiac myocytes (Huss, Levy et al. 2001)

                                    2)  affects heterodimeric parter activation of their targets

                                                a)  stop PPARa activation of responsive genes (Huss, Levy et al. 2001)

                                    3)  not related to the increase in HIF-1, because RXRa expression decrease much slower, at 12 and 24 hours after hypoxic exposure (Huss, Levy et al. 2001)

            2.  ligand-mediated downregulation

                        a)  RXRa is downregulated in response to ligand treatment through the ubiquitin-proteasome pathway (Huss, Levy et al. 2001)-33-34

                                    1)  additive with hypoxic conditions (Huss, Levy et al. 2001)


                        b)  regulation

                                    1)  RXRa may be regulated by cathepsin L-type protease (Nagaya, Murata et al. 1998)

                                    2)  cytoplasmic fraction contained the protease which cleaves RXRa’s N-terminal

                                                a)  in HepG2’s and JEG-3 cells

                                                b)  cathepsin L-type protease is a lysosomal enzyme

            3.  LPS

                        a)  causes a rapid dose-dependnet decrease in RXRa, b, and g mRNA levels (Beigneux, Moser et al. 2000)

                        b)  decreases RXR heterodimeric protein binding to DNA:  LXRa, PPARa, and RXRa homodimers (Beigneux, Moser et al. 2000)

E4.  Direct Modification

            1.  RXRa may be regulated by cathepsin L-type protease (Nagaya, Murata et al. 1998)

                                    a)  cytoplasmic fraction contained the protease which cleaves RXRa’s N-terminal

                                                1)  in HepG2’s and JEG-3 cells

                                                2)  cathepsin L-type protease is a lysosomal enzyme

E5.  Degradation

            1.  RXRa and RXRg are degraded via ubiquitination (Blanquart, Barbier et al. 2002)-8,9

                        a)  RXRa (Blanquart, Barbier et al. 2002)-9

            2.  stabilization by PPARa

                        a)  clofibrate treatment causes higher protein expression of RXRa in wild-type mice, but not in PPARa null mice in liver (Tanaka, Hora et al. 2003)

                                    1)  no effect on RNA levels

                                    2)  time course shows a dramatic increase in RXRa upon clofibrate feeding, but not in PPARa knockouts


F.  Ligands

F1.  Physiological Ligands

            1.  9-cis RA (Konta, Xu et al. 2001)-10 (Canan Koch, Dardashti et al. 1996)-9

            2.  retinoids – what are they used for?

                        a)  treatment of dermatological diseases such as acne and psoriasis (Canan Koch, Dardashti et al. 1996)-3

F2.  Arachidonic Acid Derivatives

            1.  arachidonic acid derivatives

                        a)  lipoxygenase products of arachidnoic acid activate RXR-controlled gene transcription (Eager 1992 63)

            2.  ligands

                        a)  LG 100754 – specific RXR:RXR antagonist, but RAR:RXR and PPAR:RXR agonist (Desvergne and Wahli 1999)-119,120

            3.  ligand generation

                        a)  two 9-cis-retinol dehydrogenases may participiate in synthesis of 9-cis-RA in vivo (Desvergne and Wahli 1999)-109,110

F3.  Chemicals

            1.  LG100268 (6-[1-(3,5,5,8-8-pentamethyl-5,6,7,8-tetrahydronaphthlen-2-yl)-cyclopropyl]-nicotinic acid)

                        a)  RXR agonist (Lala, Mukherjee et al. 1996)

                                    1)  concentration dependent activation (EC50 = 4nM)

                                    2)  promotes association with Gal4TBP (Lala, Mukherjee et al. 1996)

                        c)  RXR heterdimer agonist

                                    1)  activates the RXR/PPARa heterodimer at about 4mM max (Lala, Mukherjee et al. 1996)

                        d)  RAR/RXR heterdimer antagonist

                                    1)  cannot activate transcription (Lala, Mukherjee et al. 1996)

                                    2)  does not cause differentiation of the acute promyelocytic leukaemia cell line, NB4 (Lala, Mukherjee et al. 1996)

            4.  LG100754 – (2E,4E,6Z)-7-(3-n-propoxy-5,6,7,8-tetrahydro-5,5,8,8-tetramethylnaphthalen-2-yl)-3-methylocta-2,4,6-trienoic acid

                        a)  binds to RXR with nanomolar affinity (Lala, Mukherjee et al. 1996)-17

                        b)  cannot activate RBR homodimers

                        c)  RXR homodimer antagonist (Canan Koch, Dardashti et al. 1996; Lala, Mukherjee et al. 1996)

                                    1)  high affinity binding for the RXRs, and is a potent inhibitor of LGD1069 at all three RXR subtypes (Canan Koch, Dardashti et al. 1996)

                        d)  promotes associtation with Gal4TAF110 (Lala, Mukherjee et al. 1996)

                        e)  RXR heterodimer agonist

                                    1)  13-fold induction at 1mM for PPARa/RXR heterodimer (Lala, Mukherjee et al. 1996)

                                    2)  causes differentiation of the NB4 cell line (acute promyelocytic leukaemia) (Lala, Mukherjee et al. 1996)

                                    3)  activates RAR/RXR about 10 fold in an M2H assay even without RAR activator (Schulman, Li et al. 1997)

                                                a)  even mutated RXR AD still cause activation of RAR through some alternate means (Schulman, Li et al. 1997)

                                                b)  however, mutation of the RAR AD prevents activation through binding of this RXR ligand (Schulman, Li et al. 1997)

                        f)  doesn’t bind to other NRs

                                    1)  no competititon with all-trans RA for RAR (Schulman, Li et al. 1997)

F4.  Antagonists

            1.  HX531

                        a)  synthetic RXR antagonist (Yamauchi, Waki et al. 2001)-17

F5.  Retinoid Basic Information (not necessarily ligands)

            1.  properties

                        a)  in vivo are found associated wiwth cell membranes or bound to a retinoid binding protein

                                    1)  all-trans-retinol circulates in blood bound to serum retinol-binding protein (RBP)

                                    2)  all-trans-retinol and all-trans-retinal are bound to cellular retinol-binding proteins inside of cells (CRBP-1 and CRBP-2)

            2.  retinoid binding proteins (review article (Tan, Shaw et al. 2002))

                        a)  roles

                                    1)  more than just aqueous storage

                                    2)  associate with retinoids in the nM concentration range

G.  RXRa Knockout

            1.  RXRa  knockout (Wan, Cai et al. 2000)

                        a)  increased apoA-1 and apoC3 mRNA levels

                        b)  increased serum cholesterol and TAG levels

                        c)  no effect on basal acyl-CoA oxidase, medium chain acyl-CoA dehydrogenase, and malic enzyme mRNA levels

                                    1)  but, no longer inducible by Wy (Wan, Cai et al. 2000)

            2.  RXRa absent in adipocytes

                        a)  resistant to HF diet-induced obesity (Yamauchi, Waki et al. 2001)-50

H.  DNA Binding Sites

            1.  retinoic acid receptors bind a DR-1 (Durand, Saunders et al. 1992)

I.  Structure

            1.  gene structure

                        a)  transcribed from the opposite strand of the TR gene, with 3’ exons overlapping with TRa2, a dominant negative regulator of TR (Lazar, Hodin et al. 1989; Miyajima, Horiuchi et al. 1989; Lazar, Jones et al. 1990; Laudet, Begue et al. 1991)

            2.  AF-2 domain of RXRa is not critical for its ability to regulate other nuclear hormone receptors

                        a)  knockout of the AF-2 domain does not affect RXRa’s ability to help PPARg transactivation via PPARg ligands, or through RXRa ligands (Schulman, Shao et al. 1998)

            3.  mutations


                        a)  mutation stops RXRa homodimer-mediated induction (Schulman, Shao et al. 1998)

                        b)  mutation does not affect liganded RXRa mediated synergistic effects on liganded PPARg stimulation of target promoter (Schulman, Shao et al. 1998)

                        c)  mutation does not affect the recruitment of CBP by liganded PPARg (Schulman, Shao et al. 1998)






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