Can the association of Ciclosporine A and Methotrexate maintain remission/low disease activity induced by etanercept in early Rheumatoid Arthritis patients? Evaluation by Magnetic Resonance Imaging.

The opportunity to induce remission/low disease activity in Rheumatoid Arthritis (RA) patients has been achieved in recent years by the adoption of more sensitive diagnostic methods [Magnetic Resonance Imaging (MRI), ultrasonography] and early aggressive treatments (combination of biologics and synthetic DMARDs). On the other hand, data are still scarce and contrasting about the management of long-term remission. The aim of this preliminary study is to evaluate whether the association of Methotrexate + Ciclosporine A (MTX + CSA) therapy in early RA (eRA) patients is able to maintain remission/low disease activity and avoid structural progression, evaluated by MRI. Etanercept was suspended in patients who reached remission/low disease activity and CSA+MTX therapy was introduced (T0), all patients continued to receive MTX; at this time MRI showed mild/moderate synovitis and erosions in all the patients; 1-year after (T1), a slight reduction in mean synovitis, bone edema and total score was observed, whereas the erosion score was unchanged. The mean DAS44 remained stable from T0 to T1 and 6/7 patients maintained a low disease activity score. No side effects were reported. These results confirm the good clinical efficacy and safety of the combination therapy CSA+MTX in eRA patients and demonstrate a parallel arrest of structural damage evaluated by MRI 1-year after etanercept suspension.

A new chance to maintain remission induced by anti-TNF agents in rheumatoid arthritis patients: CYnAR study II of a 12-month follow-up.

The advent of biological therapies represented the beginning of a new era in the therapy of Rheumatoid Arthritis (RA), as demonstrated in several studies, but still many questions about their safety, especially in long term use, and correct administration time remain unanswered. Once remission is achieved, the orientation of clinicians regarding the maintenance of biological therapy or the switch to another immunosuppressive therapy is still uncertain. In our previous study 21 patients affected by RA who reached remission by the use of a combined therapy of anti-TNF drugs and methotrexate (MTX) underwent CyA-MTX combination therapy for maintaining remission state and were evaluated during a 6-month follow-up. The present study aims to investigate these data by a longer follow-up (12 months) and on a larger population. Fifty-three RA patients, with a disease duration of less than 3 years and DAS28<3.2 that reached a level of low disease activity within 6-8 months from the beginning of anti-TNF and methotrexate therapy, were enrolled in the study. By the suspension of anti-TNF therapy, patients underwent A-Cyclosporine (2-3 mg/kg/day) and methotrexate (15mg/week) therapy. DAS28, Pain VAS, Erythrosedimentation rate (ESR), C Reactive Protein (CRP) were all tested at time 0 and every 2 months after the interruption of the anti-TNF therapy and the beginning of A-Cyclosporine and methotrexate therapy, as well as liver and kidney profiles. Side effects were also recorded. Of 53 patients, 50 completed the study with a 12-month follow-up. Twenty-one (42%) patients maintained clinical parameters within low disease activity values at 12 months, while 29 (58%) patients showed an increase in DAS28 and other parameters: 16 (32%) patients at the 6-month control, 13 (26%) patients at the 12-month control. Our data show that 42% of the patients undergoing A-Cyclosporin and Methotrexate therapy maintained low disease activity parameters of rheumatoid arthritis, obtained after 6-8 months of anti-TNF therapy. Further studies on larger populations are necessary in order to confirm such results and identify predictor factors for different responses.

Cyclosporine A in the long-term management of systemic lupus erythematosus.

To retrospectively evaluate safety and efficacy of long-term treatment with Cyclosporine A (CSA) in patients with systemic lupus erythematosus (SLE) poorly responsive to treatment with corticosteroids (CCS) and/or conventional disease-modifying anti-rheumatic drugs (DMARDs), SLE patients who had received CSA-based induction and maintenance regimens according to disease activity were recorded. Efficacy was assessed using the SLE Disease Activity Index (SLEDAI) and laboratory analyses. Forty SLE patients (including 18 with lupus nephritis, 11 with neurological involvement and 7 with overlap syndromes (4 Sjögren's syndrome, 2 myasthenia gravis and 1 Behçet's disease) were recorded. According to baseline SLEDAI, 30 patients had severe and 10 moderate SLE. Mean SLEDAI scores and relevant laboratory values significantly reduced from baseline (22∓10 vs 5∓6; P < 0.002) during the follow-up period (8∓2 years; range 1-15). Twenty-three (57.5 percent) patients achieved excellent (improvement in the range 70-100 percent) response to treatment (10 of whom were subsequently maintained on CSA monotherapy), 14 (35 percent) had good-fair (improvement in the range 25-69 percent) response and 3 (7.5 percent) had to interrupt therapy (including CSA) for disease worsening. Mild and transient adverse events occurred in 15 (37 percent) patients, including hypertrichosis (17.5 percent), gum hypertrophy (17.5 percent) hypertension (12.5 percent), abdominal pain (7.5 percent), and dyslipidemia (5 percent), but treatment interruption was not required. Low-dose CSA together with other drugs is effective to induce, or as monotherapy to maintain, long-term (at least 2 years) remission, and is generally well tolerated in patients with moderate or severe SLE poorly responsive to CCS and/or conventional DMARDs. Furthermore, the favourable effect of CSA treatment may allow to spare more cytotoxic drugs.

Influenza vaccine administration in rheumatoid arthritis patients under treatment with TNFalpha blockers: safety and immunogenicity.

Twenty-eight patients with low-moderate, stable rheumatoid arthritis (RA), under treatment with tumor necrosis factor (TNF) alpha blockers, were immunized at least once with non-adjuvanted trivalent influenza vaccine during three consecutive influenza seasons. Antibodies toward A influenza antigens significantly increased and reached protective levels, still detectable 6 months after vaccination, both in RA patients and healthy controls. Response to B antigen instead was only observed from the second year for healthy controls and in the third year for patients. No significant difference in disease activity and anti-nuclear antibodies was observed as a consequence of vaccine administration, whereas T regulatory cells showed a significant increase 30 days after immunization in RA patients. This study confirms safety of influenza vaccine administration in RA patients treated with TNFalpha blockers. The cohort follow-up revealed the overcoming of poor B vaccine antigen immunogenicity via repeated vaccinations. Finally, protective antibody response was still observed 6 months after vaccination.

Can Cyclosporine-A associated to methotrexate maintain remission induced by anti-TNF agents in rheumatoid arthritis patients? (Cynar pilot study).

Biological therapies, such as etanercept, adalimumab and infliximab, have demonstrated good efficacy in inducing rheumatoid arthritis to low disease activity levels. Nevertheless, their cost, as well as the related risk of side effects, especially in long-term therapies, are still high. Furthermore, there is a good deal of evidence proving loss of efficacy of such therapies in the long term, often necessitating the shift from one specific anti-TNF biological treatment to another. There are also other open debates on the amount of time a patient should undergo an anti-TNF therapy, on the possibility of inducing a complete remission in early arthritis and, once remission or low disease activity is obtained, on the possibility of interrupting the anti-TNF-based therapy. In this study we investigated whether A-Cyclosporin and Methotrexate association may be effective in maintaining low disease activity obtained by anti-TNF therapies. Twenty-three rheumatoid arthritis-affected patients, whose diagnosis was made according to ACR criteria, with a disease duration of less than 3 years, and DAS28<3.2 that reached a level of low disease activity within 6-8 months from beginning anti-TNF and Methotrexate therapy, were enrolled in the study. After the suspension of anti-TNF therapy, patients were started on A-Cyclosporine (2-3 mg/kg/day) and Methotrexate (15mg/week) therapy. DAS28, Pain VAS, Erythrosedimentation Rate (ESR), and C Reactive Protein (CRP) were all tested at time 0 and at 6 months, as well as liver and kidney profiles, after the interruption of the anti-TNF therapy and the beginning of A-Cyclosporine and Methotrexate therapy. Side effects were also recorded. Of 23 patients undergoing the A-Cyclosporin and Methotrexate therapy for maintaining low disease activity in rheumatoid arthritis obtained by 6-8 months of anti-TNF therapy, 21 completed the study with a 6 month follow-up. Thirteen patients maintained clinical parameters within low disease activity values, while 8 patients showed an increase in DAS28 and other parameters. Only two patients showed an increase in blood pressure that was diagnosed after two months from the beginning of the A-Cyclosporin and Methotrexate therapy. The reduction in the dosage of A-Cyclosporin from 3mg/kg/day to 2mg/kg/day caused a slow normalization of blood pressure values. Our data seem to suggest that more than half of the patients undergoing A-Cyclosporin and Methotrexate therapy seemed to maintain low disease activity parameters of rheumatoid arthritis, obtained after 6-8 months of anti-TNF therapy. Further studies on larger populations are necessary in order to confirm such results and identify predictor factors for different responses.

Enzymes involved in arachidonic acid release in adrenal and Leydig cells.

Stimulation of receptors and subsequent signal transduction results in the activation of arachidonic acid (AA) release. Once AA is released from phospholipids or others esters, it may be metabolized via the cycloxygenase or the lipoxygenase pathways. How the cells drive AA to these pathways is not elucidated yet. It is reasonable to speculate that each pathway will have different sources of free AA triggered by different signal transduction pathways. Several reports have shown that AA and its lipoxygenase-catalyzed metabolites play essential roles in the regulation of steroidogenesis by influencing cholesterol transport from the outer to the inner mitochondrial membrane, the rate-limiting step in steroid hormone biosynthesis. Signals that stimulate steroidogenesis also cause the release of AA from phospholipids or other esters by mechanisms that are not fully understood. This review focuses on the enzymes of AA release that impact on steroidogenesis.

Membrane methylation in isolated rat testis interstitial cells unmasks functional luteinizing hormone receptors.

Treatment of intact isolated rat testis interstitial cells with S-adenosylmethionine as methyl donor, increases substantially the number of LH human CG receptors (100-200%) without modifying the equilibrium dissociation constant. The increase in binding capacity was associated with an augmentation in the sensitivity of the rat testis interstitial cells to produce testosterone in response to LH, suggesting a functional role of the unmasked receptors. The amount of S-adenosylmethionine necessary to obtain an increase in LH binding capacity and preserve cell viability was 25-50 micrograms/ml per 1.6 X 10(7) cells. 10 mM MgCl2 in addition to the Mg2+ present in the medium was necessary to maintain cell viability. 3H-labelled methyl groups were incorporated mainly into the lipid fraction (208 fmol/10(6) cells) when 3H-S-adenosylmethionine was incubated with the cells for 2 h at 30 degrees C. Our results are consistent with the conclusion that early action of LH may involve an activation of methyltransferase activity, phospholipid methylation, an increase in LH binding capacity and an increase in receptor function.

Activation of a thioesterase specific for very-long-chain fatty acids by adrenergic agonists in perfused hearts.

We have recently described an acyl-CoA thioesterase specific for very-long-chain fatty acids, named ARTISt, that regulates steroidogenesis through the release of arachidonic acid in adrenal zona fasciculata cells. In this paper we demonstrate the presence of the protein as a 43 kDa band and its mRNA in cardiac tissue. The activity of the protein was measured using an heterologous cell-free assay in which it is recombined with adrenal microsomes and mitochondria to activate mitochondrial steroidogenesis. Isoproterenol and phenylephrine activate the enzyme in a dose-dependent manner (10(-10)-10(-6) M). Both propranolol (10(-5) M) and prazosin (10(-5) M) block the action of isoproterenol and phenylephrine respectively. Antipeptide antibodies against the serine lipase motif of the protein and the Cys residue present in the catalytic domain also block the activity of the protein. Taken together, our results confirm the presence of ARTISt in heart and provide evidence for a catecholamine-activated regulatory pathway of the enzyme in that tissue.

Site of action of proteinases in the activation of steroidogenesis in rat adrenal gland.

We have investigated the effect of the proteinase inhibitors 1,10-phenantroline (OP) and phenylmethylsulfonyl fluoride (PMSF) on steroidogenesis in rat adrenal cortex. Both PMSF and OP inhibited adrenocorticotropin (ACTH)- and 8-Br cAMP-induced stimulation of corticosterone synthesis. On the contrary, arachidonic acid-induced stimulation of corticosterone synthesis was only slightly inhibited by PMSF and unchanged by OP. Intra- and extracellular cAMP levels were determined by radioimmunoassay. While PMSF did not affect neither the intra- nor the extracellular cAMP levels, OP decreased the intra- and extracellular levels of unstimulated as well as ACTH-stimulated cells. The site of action of the proteinase inhibitors was also studied by recombination of mitochondria with the different subcellular fractions in vitro. Addition of PMSF abolished the stimulation achieved by in vitro activation of cytosol by cAMP and PKA. On the other hand, OP completely inhibited the activation of mitochondria. Our results provide evidence for the involvement of proteinases in ACTH-induced stimulation of steroidogenesis in adrenal cortex both prior to the release of arachidonic acid and at the level of cholesterol transport from the outer to the inner mitochondrial membrane.

Penetrance and clinical manifestations of non-hotspot germline RET mutation, C630R, in a family with medullary thyroid carcinoma.

Germline mutations in specific hot spot-codons of the RET proto-oncogene are associated with multiple endocrine neoplasia type 2 (MEN 2). Clinical RET gene testing has been routine for the last 10 years in some countries. In Argentina, RET testing excluding MEN 2B was always reported with a mutation at codon 634, with one exception: we described a novel mutation T > C transition at codon 630 (C630R), the family to which we extend the study in the present report. This family comprised 29 members in four generations including 6 individuals affected with medullary thyroid cancer (MTC), positive for the C630R mutation and normal adrenaline/ noradrenaline and ionized calcium/parathyroid hormone levels. Two asymptomatic mutation carriers aged 5 and 11 years underwent total thyroidectomy. The histopathologic examination showed C-cell hyperplasia and microcarcinoma foci, while preoperative basal calcitonins were normal for both. Our report emphasizes the importance of testing for non-hot spot RET mutations in apparently mutation negative MEN 2 families. Furthermore, it would appear that C630R mirrors C634R in penetrance (100% in this family) and in early age of onset of MTC, although paradoxically, no pheochromocytomas and hyperparathyroidism have developed. In addition to recommending RET testing before 5 years of age; we also can postulate that codon 630 may be the key point along the extracellular domain, important in the tissue-specific penetrance.

Luteinizing hormone triggers two opposite regulatory pathways through an initial common event, receptor aggregation.

LH receptor internalization was studied with an antireceptor monoclonal antibody (aLHR) which induces Leydig cells to produce testosterone. To follow receptor-mediated aLHR internalization, cells were incubated with aLHR at 10 C for 3 h to generate an aLHR complex; this was followed by a second incubation with fluorescent labeled antimouse immunoglobulin at 34 C, a temperature which allows internalization. Within 15 min at 34 C, cytoplasmic fluorescent staining was detectable; this staining was strongly visible after 60 min. At no time was nuclear staining observable. Employing such an approach, it has also been possible to follow the fate of unoccupied receptors when cells are stimulated with a submaximal dose of LH. The results show that LH interactions with 20% of its receptors produces microaggregation, patching, capping, and internalization of free receptor sites. The results further demonstrate that cells with receptors in the state of capping are less sensitive to a second LH stimulation, suggesting that in this state receptors are no longer coupled to the adenylate cyclase system.

Luteinizing hormone triggers a molecular association between its receptor and the major histocompatibility complex class I antigen to produce cell activation.

We studied the involvement of major histocompatibility (MHC) class I antigens on the mechanism of LH/hCG receptor activation. For this purpose we investigated the effects of anti-MHC class I antibodies on hormone-receptor interaction, signal transduction, and MHC class I antigen-receptor interaction. Monoclonal antibodies against MHC class I antigen were able to stimulate testosterone production in mouse Leydig cells with the same potency as LH. This biological effect depends on the concentration of antibody used and could be abolished by a LH antagonist. There is a perfect parallelism, for each monoclonal antibody, between the specificity for a particular haplotype and the response of the target cells from the strains carrying such a haplotype. The same antibodies were able to precipitate the soluble LH/hCG receptors, as both a hormone-receptor complex and a free receptor. The results suggest that bound hormone triggers an association of the MHC class I antigen with the LH/hCG receptor, resulting in activation of the target cell.

Effects of L-arginine in rat adrenal cells: involvement of nitric oxide synthase.

The effects of L-arginine on corticosterone production, cGMP, and nitrite levels were examined in zona fasciculata adrenal cells. L-Arginine significantly decreased both basal and ACTH-stimulated corticosterone production. This effect was still evident when steroidogenesis was induced by 8-bromo-cAMP and 22(R)-hydroxycholesterol, but not in the presence of exogenously added pregnenolone. L-Arginine increased cGMP and nitrite levels,; these effects were blocked by the nitric oxide synthase inhibitor, N(G)-nitro-L-arginine methyl-ester. Transport of L-[3H]arginine was rapid, saturable, and monophasic, with an apparent Km of 163+/-14 microM and a maximum velocity of 53+/-6 pmol/min x 10(5) cells. The basic amino acids L-lysine and L-ornithine, but not D-arginine or the nitric oxide synthase inhibitors N(G)-nitro-L-arginine methyl-ester and N(G)-nitro-L-arginine, impaired L-arginine uptake. Taken together, these results suggest that steroidogenesis in zona fasciculata adrenal cells may be negatively modulated by L-arginine-derived nitric oxide.

The effect of castration and testosterone replacement on specific proteins and androgen levels of the rat epididymis.

The normal weight increase of the epididymis during sexual maturation and its maintenance through adulthood were found to be dependent on the provision of androgens. Binding of [3H]dihydrotestosterone (DHT) to the epididymal 8S cytoplasmic receptor gradually decreased after castration to become undetectable after 25 days. Binding to the androgen binding protein (ABP) was absent 4 days after castration and was not reinduced by 3 weeks of testosterone (T) administration. Unilateral castration for periods of up to 27 days showed the disappearance of ABP with preservation of the 8S receptor on the castrated side, indicating a testicular source for ABP and the epididymal origin of the 8S receptor. The tissue concentrations of T and DHT in the epididymis became undetectable 30 days after castration and were restored to normal values by administration of testosterone in large doses (1.5 mg/100 g BW). Similar results were obtained in rats castrated at 10 days of age and injected with testosterone until 60 days old. The ratio DHT/T was depressed in the castrate and increased with testosterone treatment. The protein content of the epididymis (mg of protein/g wet weight) was also found to be influenced by androgens. Our results show evidence of some mechanisms involved in the trophic effect of androgens upon the epididymis and suggest the possible androgenic control of epididymal 5alpha-reductase activity. They also indicate that a testicular factor is required for the maintenance of the 8S cytoplasmic androgen receptor. It is not known whether this factor is testosterone or some other testicular secretion.

Comparison between bioactive and immunoactive luteinizing hormone (LH) in ovariectomized streptozotocin-induced diabetic rats: response to LH-releasing hormone.

The effect of streptozotocin-induced diabetes on circulating levels of immunoactive LH (I-LH) and bioactive LH (B-LH) was investigated. LH was measured in adult ovariectomized (OVX) rats before and after acute LHRH administration, with or without estradiol benzoate (Eb) treatment (10 micrograms, 48 and 24 h before experiments). I-LH and B-LH were measured in the same samples by RIA and the rat interstitial cell testosterone assay, respectively. OVX diabetic animals showed a significant reduction in both I-LH (63%) and B-LH (73%). Treatment with Eb induced a decrease in basal I-LH and B-LH levels in all experimental groups (50%). These values were normalized after insulin therapy. No alterations in the pituitary responsiveness to LHRH were detected when I-LH levels were determined. However, B-LH levels assayed after LHRH stimulation were significantly decreased in diabetic animals. Insulin treatment was unable to restore this response. The effect of Eb treatment on these parameters was also tested. In these conditions LHRH injections induced similar increases in serum I-LH and B-LH in both diabetic and control rats. These results indicate that, in diabetic OVX rats, basal and LHRH-induced LH has a reduced bioactivity, but this reduction is reversed by Eb treatment. This might indicate that the major defect lies in the ovary rather than at the pituitary level, supporting the notion of an important role of the steroid milieu on the B-LH modulation.

Lipoxygenase products as common intermediates in cyclic AMP-dependent and -independent adrenal steroidogenesis in rats.

Aldosterone secretion from adrenal glomerulosa cells can be stimulated by angiotensin II (AII), extracellular potassium and ACTH. Mitochondria from these cells respond to intracellular factors generated by AII (cyclic AMP (cAMP)-independent steroidogenesis) and ACTH (cAMP-dependent steroidogenesis), suggesting that the two-signal-transduction mechanisms are linked by a common intermediate. We have evaluated this hypothesis by stimulating mitochondria from the unstimulated zona glomerulosa with a subcellular post-mitochondrial fraction (PMF) obtained from the zona glomerulosa after stimulation with AII or from the fasciculata gland after stimulation with ACTH; the subcellular fractions were also tested on mitochondria from fasciculata cells. PMFs obtained after incubation of adrenal zona glomerulosa with or without AII (0.1 microM) or ACTH (0.1 nM) were able to increase net progesterone synthesis 4.5-fold in mitochondria isolated from unstimulated rat zona glomerulosa. AII-pretreated PMFs from the zona glomerulosa also stimulated steroidogenesis by mitochondria from zona fasciculata cells. Separate experiments showed that inhibitors of arachidonic acid release and metabolism (bromophenacyl bromide, nordihydroguaiaretic acid, caffeic acid or esculetin) blocked corticosterone production in fasciculata cells stimulated with ACTH, suggesting that arachidonic acid could be the common intermediate in the actions of AII and ACTH on steroid synthesis. Evidence to support this concept was obtained from experiments in which the formation of an activated PMF by treatment of zona fasciculata with ACTH was blocked by the presence of the same inhibitors. Moreover, the inhibitory effects of these substances on PMF activation by ACTH were overcome by exogenous arachidonic acid and, in addition, arachidonic acid release was stimulated by ACTH.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation of an individual cell with peptide hormone in a prescribed region of its plasma membrane results in a compartmentalized cyclic AMP-dependent protein kinase response.

This work describes the stimulation by a peptide hormone of an individual cell in a prescribed region of its plasma membrane. When Leydig cells were stimulated via a section of membrane tightly sealed to an electrode containing LH, a very localized area exhibited the morphological change known as 'rounding up', which is a cyclic AMP-dependent protein kinase-mediated response. This localized stimulation did not produce a wider response through intracellular, intermembranous or extracellular signals. Each individual cell responded to peptide stimulation gradually, with an increase over time and with dose. In contrast, when the stimulation was accomplished using a non-hydrolysable cyclic AMP analogue in the patch electrode, a general response throughout an individual cell was produced. Locally stimulated peptide hormone receptors, adenylate cyclases and cyclic AMP-dependent protein kinases appear to be closely associated so that second messenger production and the effects it mediates are compartmentalized.

Effect of nitric oxide on rat adrenal zona fasciculata steroidogenesis.

The present study was designed to investigate the role of nitric oxide (NO) in the regulation of adrenocortical function. Different NO donors, such as sodium nitroprusside (SNP), S-nitroso-L-acetyl penicillamine, diethylamine/NO complex sodium salt and diethylenetriamine NO adduct, significantly decreased corticosterone production both in unstimulated and in corticotropin-stimulated zone fasciculata adrenal cells, in a dose-dependent manner. The effect of SNP was reversed by ferrous hemoglobin. A selective inhibitor of NO synthase, L-NG-nitro-arginine significantly increased corticosterone secretion. The effect of SNP was not mediated by cGMP as permeable cGMP analogs did not reproduce its inhibitory effect. SNP significantly inhibited the steroidogenesis stimulated by 8Br-cAMP and 22(R)-OH-cholesterol, but was ineffective when corticosterone was produced in the presence of exogenously added pregnenolone. Moreover, the conversion of [3H]cholesterol to [3H]pregnenolone and the production of pregnenolone or progesterone (assessed by RIA) were significantly decreased by SNP. Taken together, these results suggest that NO may be a negative modulator of adrenal zona fasciculata steroidogenesis.

Adrenocorticotropin induces heme oxygenase-1 expression in adrenal cells.

Heme oxygenase (HO) catalyzes the first and rate-controlling step of heme catabolism into biliverdin, iron and carbon monoxide. Three isoforms of HO have been identified so far: the inducible HO-1 and the constitutive HO-2 and HO-3. Both HO-1 and HO-2 were expressed in zona fasciculata (ZF) adrenal cells and in a mouse adrenocortical cell line (Y1). HO-1 but not HO-2 expression was upregulated by adrenocorticotropic hormone (ACTH) and accumulation of HO-1 protein correlated with an increase in HO activity in Y1 cells. ACTH induced HO-1 expression in a time- and dose-dependent manner with a maximum after 5 h of treatment and a threshold concentration of 0.1 mIU/ml. Actinomycin D and cycloheximide completely blocked the effect of ACTH on HO-1 mRNA expression whereas mRNA stability was not affected by ACTH. Permeable analogs of cAMP mimicked the effect of ACTH on HO-1 expression and ACTH induction was prevented by the protein kinase A (PKA) inhibitor H89. Steroid production was significantly increased when both HO-1 and HO-2 activities were inhibited by Sn-protoporphyrin IX (SnPPIX). The lipid peroxidation and increase in carbonyl content triggered by hydrogen peroxide was prevented by treatment of Y1 cells with bilirubin and ACTH.

LH/chorionic gonadotropin signaling pathway involves protein tyrosine phosphatase activity downstream of protein kinase A activation: evidence of an obligatory step in steroid production by Leydig cells.

Our recent reports indicate that protein tyrosine phosphorylation is an obligatory component of the mechanism of action of ACTH in its stimulatory action of corticosteroid production in adrenal zona fasciculata (ZF). The role of protein tyrosine phosphatase (PTP) activity in the regulation of steroidogenesis by LH/chorionic gonadotropin (CG) was tested using cell-permeable PTP inhibitors. Thus, PTP inhibition blocks LH- and 8-bromo-cAMP-stimulated testosterone production by Leydig cells without affecting 22(R)OH-cholesterol-supported steroidogenesis, similar results to those obtained in the adrenal ZF/ACTH system, leading us to propose that PTP action is an obligatory and common step in the cascade triggered by both hormones. Then, we continued the study testing whether LH modulates PTP activity in MA-10 cells, a Leydig cell line. In this regard, we observed by an in-gel PTP assay two PTPs of 110 and 50 kDa that are activated by hormone and 8-bromo-cAMP activation of the cells. Moreover, there is a transient increase by the second messenger in total PTP activity that correlates with the higher activity displayed by the 110 and 50 kDa proteins in the in-gel assay. In accordance with these results, analysis of tyrosine phosphorylated proteins showed the LH-induced dephosphorylation of proteins of 120, 68 and 50 kDa. The results of this study indicate that PTPs play an important role in the regulation of Leydig cell functions and that there exists a cross talk between serine/threonine phosphorylation and tyrosine dephosphorylation mediated by hormone-activated cAMP-dependent protein kinase and PTPs. These results are the first evidence of PTP having a role in LH/CG-stimulated steroidogenesis.