María J. Toro

Ligand stabilization of CXCR4/δ-opioid receptor heterodimers reveals a mechanism for immune response regulation

The CXCR4 chemokine receptor and the delta opioid receptor (DOR) are pertussis toxin‐sensitive G protein‐coupled receptors (GPCR). Both are widely distributed in brain tissues and immune cells, and have key roles in inflammation processes and in pain sensation on proximal nerve endings. We show that in immune cells expressing CXCR4 and DOR, simultaneous addition of their ligands CXCL12 and [D‐Pen2, D‐Pen5]enkephalin does not trigger receptor function. This treatment does not affect ligand binding or receptor expression, nor does it promote heterologous desensitization. Our data indicate that CXCR4 and DOR form heterodimeric complexes that are dynamically regulated by the ligands. This is compatible with a model in which GPCR oligomerization leads to suppression of signaling, promoting a dominant negative effect. Knockdown of CXCR4 and DOR signaling by heterodimerization might have repercussions on physiological and pathological processes such as inflammation, pain sensation and HIV‐1 infection.

Lovastatin induces apoptosis of spontaneously immortalized rat brain neuroblasts: involvement of nonsterol isoprenoid biosynthesis inhibition.

We have examined the effects of lovastatin and pravastatin (competitive HMG-CoA reductase inhibitors) on the growth and survival of rat brain neuroblasts. Lovastatin, but not pravastatin, suppressed cell growth by inducing apoptosis of neuroblasts in a dose- and time-dependent manner. Apoptosis was accompanied by a decrease in both Bcl-2 and Bcl-xL protein levels, suggesting that changes in the expression of these genes may contribute to apoptosis following lovastatin treatment. Lovastatin treatment was also associated with decreased prenylation of both Ras and Rho A proteins whereas Rac 1 geranylgeranylation was not affected. Lovastatin effects were fully prevented by mevalonate. The present data suggest that lovastatin induces apoptosis of rat brain neuroblasts by its capacity to decrease the prenylation of specific proteins involved in signal transduction pathways that control growth and survival of neuronal cells.

RhoA and p38 MAPK mediate apoptosis induced by cellular cholesterol depletion

Cholesterol is essential for cell viability, and homeostasis of cellular cholesterol is crucial to various cell functions. Here we examined the effect of cholesterol depletion on apoptosis and the mechanisms underlying this effect in NIH3T3 cells. We show that chronic cholesterol depletion achieved with lipoprotein-deficient serum (LPDS) and 25-hydroxycholesterol (25-HC) treatment resulted in a significant increase in cellular apoptosis and caspase-3 activation. This effect is not due to a deficiency of nonsterol isoprenoids, intermediate metabolites of the cholesterol biosynthetic pathway, but rather to low cholesterol levels, since addition of cholesterol together with LPDS and 25-HC nearly abolished apoptosis, whereas addition of farnesyl pyrophosphate or geranylgeranyl-pyrophosphate did not reverse the cell viability loss induced by LPDS plus 25-HC treatment. These effects were accompanied by an increase in ERK, JNK and p38 MAPK activity. However, only the inhibition of p38 MAPK with the specific inhibitor SB203580 or the overexpression of a kinase defective MKK6 resulted in a significant decrease in apoptosis and caspase-3 cleavage induced by cholesterol depletion. Furthermore, LPDS plus 25-HC increased RhoA activity, and this effect was reversed by addition of exogenous cholesterol. Finally, overexpression of the dominant negative N19RhoA inhibited p38 MAPK phosphorylation and apoptosis induced by low cholesterol levels. Together, our results demonstrate that cholesterol depletion induces apoptosis through a RhoA- and p38 MAPK-dependent mechanism.

Mutagenesis in the switch IV of the helical domain of the human Gsα reduces its GDP/GTP exchange rate

The Gα subunits of heterotrimeric G proteins are constituted by a conserved GTPase “Ras‐like” domain (RasD) and by a unique α‐helical domain (HD). Upon GTP binding, four regions, called switch I, II, III, and IV, have been identified as undergoing structural changes. Switch I, II, and III are located in RasD and switch IV in HD. All Gα known functions, such as GTPase activity and receptor, effector, and Gβγ interaction sites have been found to be localized in RasD, but little is known about the role of HD and its switch IV region. Through the construction of chimeras between human and Xenopus Gsα we have previously identified a HD region, encompassing helices αA, αB, and αC, that was responsible for the observed functional differences in their capacity to activate adenylyl cyclase (Antonelli et al. [1994]: FEBS Lett 340:249–254). Since switch IV is located within this region and contains most of the nonconservative amino acid differences between both Gsα proteins, in the present work we constructed two human Gsα mutant proteins in which we have changed four and five switch IV residues for the ones present in the Xenopus protein. Mutants M15 (hGsααS133N, M135P, P138K, P143S) and M17 (hGsααS133N, M135P, V137Y, P138K, P143S) were expressed in Escherichia coli, purified, and characterized by their ability to bind GTPγS, dissociate GDP, hydrolyze GTP, and activate adenylyl cyclase. A decreased rate of GDP release, GTPγS binding, and GTP hydrolysis was observed for both mutants, M17 having considerably slower kinetics than M15 for all functions tested. Reconstituted adenylyl cyclase activity with both mutants showed normal activation in the presence of AlF4−, but a decreased activation with GTPγS, which is consistent with the lower GDP dissociating rate they displayed. These data provide new evidence on the role that HD is playing in modulating the GDP/GTP exchange of the Gsα subunit. J. Cell. Biochem. 76:368–375, 2000.

Lovastatin decreases prolactin and growth hormone gene expression in GH4C1 cells through a cAMP dependent mechanism.

The heterotrimeric G protein Gs couples several surface ligand receptors to cAMP production, as well as to both growth hormone (GH) and prolactin (PRL) gene expression in pituitary and GH cells. It has been shown that constitutively active alpha s stimulates transient expression of both PRL- and GH- chloramphenicol acetyl transferase (CAT) constructions, which indicates that both the PRL and GH promoter regions are under the influence of signal pathways mediated by alpha s. We have previously shown that the cholesterol lowering drug lovastatin decreases both the amount of G alpha s subunit in the membrane and the adenylyl cyclase activity in GH4C1 cells. Thus, we tried to verify whether that decrease in alpha s levels could affect PRL and GH secretion, as well as the expression of PRL- and GH-CAT constructions. Since the regulation of these two genes is dependent on the pituitary specific transcription factor Pit-1, the effect of lovastatin on the expression of Pit-1-CAT constructions was also studied. Our results show that lovastatin decreased the basal expression of these three cAMP-responsive genes in GH4C1 cells, being partially reversed by the addition of mevalonate to the culture medium. This effect of lovastatin on the promoter activities of the transfected constructions was also observed in PRL and GH secretion to the medium, suggesting that this drug produces similar changes in the endogenous promoters of both hormones. Moreover, the presence of lovastatin did not prevent the response to the cAMP activator forskolin, indicating that the main effect of this drug could be exerted through upstream adenylyl cyclase. In conclusion, our data indicate that lovastatin decreases the basal expression of Pit-1 and consequently of both GH and PRL genes through a mechanism probably mediated by the decrease of G alpha s levels in the cell membrane. Taken together, these results suggest that the activity of membrane heterotrimeric G proteins regulates the basal transcription of specific cellular genes in GH4C1 cells. Moreover the effects of lovastatin may be taken into account in the study of constitutively endocrine disorders associated with an increased secretion of either PRL or GH.

Effects of lovastatin on adenylyl cyclase activity and G proteins in GH4C1 cells

We studied the effect of lovastatin, a cholesterol lowering drug, on the basal state of G‐proteins in GH4C1 cells. Our data show that the addition of lovastatin markedly decreased the amount of the α‐subunits of the Gs and Gi‐proteins in the plasma membrane. The decrease of αs was correlated with a decrease in adenylyl cyclase activity, and both effects were reverted by the presence of mevalonate. As the attachment of G protein subunits to the membrane is dependent on γ‐subunit prenylation, we assume that the mechanism through which lovastatin exerts its effects on G‐proteins is the lack of mevalonate for the synthesis of prenyl residues. In conclusion, our data indicate that some of the effects of lovastatin are mediated through changes in the basal state of G‐protein in the membrane and consequently on adenylyl cyclase activity.

Oncogenic Ras, but not V600EB-RAF, protects from cholesterol depletion-induced apoptosis through the PI3K/AKT pathway in colorectal cancer cells

Cholesterol is necessary for proliferation and survival of transformed cells. Here we analyse the effect of cholesterol depletion on apoptosis and the mechanisms underlying this effect in colorectal cancer cells carrying oncogenic Ras or (V600E)B-RAF mutations. We show that chronic cholesterol depletion achieved with lipoprotein-deficient serum (LPDS) and 25-hydroxycholesterol (25-HC) treatment results in a significant increase in apoptosis in HT-29 and Colo-205 cells containing the (V600E)B-RAF mutation, but not in HCT-116 and LoVo cells harbouring the (G13D)Ras mutation, or BE cells, which possess two mutations, (G13D)Ras and (G463V)B-RAF. We also demonstrate that oncogenic Ras protects from apoptosis induced by cholesterol depletion through constitutive activation of the phosphatidylinositol-3 kinase (PI3K)/AKT pathway. The specific activation of the PI3K/AKT pathway by overexpression of the (V12)RasC40 mutant or a constitutively active AKT decreases the LPDS plus 25-HC-induced apoptosis in HT-29 cells, whereas PI3K inhibition or abrogation of AKT expression renders HCT-116 sensitive to cholesterol depletion-induced apoptosis. Moreover, our data show that LPDS plus 25-HC increases the activity of c-Jun N-terminal kinase proteins only in HT-29 cells and that the inhibition of this kinase blocks the apoptosis induced by LPDS plus 25-HC. Finally, we demonstrate that AKT hyperactivation by oncogenic Ras protects from apoptosis, preventing the activation of c-Jun N-terminal kinase by cholesterol depletion. Thus, our data demonstrate that low levels of cholesterol induce apoptosis in colorectal cancer cells without oncogenic Ras mutations. These results reveal a novel molecular characteristic of colon tumours containing Ras or B-RAF mutations and should help in defining new targets for cancer therapy.

Effect of mevalonate availability on the association of G-protein α-subunits with the plasma membrane in GH4C1 cells

We show that the levels and activity of the α‐subunits of Gs and Gi proteins in plasma membrane of GH4C1 cells are regulated by the availability of mevalonate (MVA), and not by changes in cholesterol cell content. Changes in the levels of MVA, induced by modulation of 3‐hydroxy‐3‐methylglutaryl coenzyme A (HMG‐CoA) reductase, determine the amount of both membrane‐bound Gα‐subunits, which correlated with the activity of their effector adenylyl cyclase. Lipoprotein deficient serum (LPDS) decreases cholesterol content and increases both HMG‐CoA reductase activity and Gα‐subunits in the membrane. Cholesterol and 25‐hydroxycholesterol (25‐HC) each repress HMG‐CoA reductase and diminish Gα‐subunit levels. However, while cholesterol cell content is also decreased by 25‐HC, exogenous cholesterol increases it. In addition, the decrease of both Gα‐subunits is reversed by the presence of MVA. This regulation appears to be mediated by nonsterol products generated from MVA. We assume that the first is the prenylation of the γ‐subunits, since the attachment of Gα‐subunits to the membrane is dependent on this modification. However, as neither of our treatments completely abolished protein prenylation, we conclude that another MVA derivative is required in addition to prenyl residues to the presence and activity of α‐subunits in the membrane.

Denervation-induced supersensitivity to forskolin and pinacidil is not related to changes in the adenylate cyclase transduction pathway in the rabbit femoral artery

1. The present study investigates whether chemical sympathectomy compromises the relaxation of the rabbit femoral artery precontracted with serotonin. The vasodilating agents promoted cyclic adenosine monophosphate (cAMP) accumulation or opening of the potassium channels. The effect of denervation on the adenylyl cyclase transduction pathway was also studied. 2. 6-Hydroxydopamine treatment did not impair the relaxation to adenosine, 8-bromoadenosine-cAMP (a membrane-permeable analog of cAMP) and 3-isobutyl-1-methylxanthine (a cAMP phosphodiesterase inhibitor). Moreover, denervation enhanced the relaxation to forskolin (a direct Gs-type protein activator) and pinacidil (a potassium channel opener). 3. Denervation modified neither adenosine diphosphate ribosylation of Gs- and Gi-proteins nor adenylyl cyclase activity.