Hugo A. Armelin

Unmasking a growth-promoting effect of the adrenocorticotropic hormone in Y1 mouse adrenocortical tumor cells.

The adrenocorticotropic hormone (ACTH) inhibits the growth of Y1 mouse adrenocortical tumor cells as well as normal adrenocortical cells in culture but stimulates adrenocortical cell growth in vivo. In this study, we investigated this paradoxical effect of ACTH on cell proliferation in Y1 adrenal cells and have unmasked a growth-promoting effect of the hormone. Y1 cells were arrested in the G1 phase of the cell cycle by serum starvation and monitored for progression through S phase by measuring [3H]thymidine incorporation into DNA and by measuring the number of nuclei labeled with bromodeoxyuridine. Y1 cells were stimulated to progress through S phase and to divide after a brief pulse of ACTH (up to 2 h). This effect of ACTH appeared to be cAMP independent, since ACTH also induced cell cycle progression in Kin-8, a Y1 mutant with defective cAMP-dependent protein kinase activity. The growth-promoting effect of ACTH in Y1 was preceded by the rapid activation of p44 and p42 mitogen-activated protein kinases and by the accumulation of c-FOS protein. In contrast, continuous treatment with ACTH (14 h) inhibited cell cycle progression in Y1 cells by a cAMP-dependent pathway. The inhibitory effect of ACTH mapped to the midpoint of G1. Together, the results demonstrate a dual effect of ACTH on cell cycle progress, a cAMP-independent growth-promoting effect early in G1possibly mediated by mitogen-activated protein kinase and c-FOS, and a cAMP-dependent inhibitory effect at mid-G1. It is suggested that the growth-inhibitory effect of ACTH at mid-G1 represents an ACTH-regulated check point that limits cell cycle progression.

ACTH receptor: Ectopic expression, activity and signaling

Failure in obtaining expression of functional adrenocorticotropic hormone receptor (ACTHR, or melanocortin 2 receptor, MC2R) in non-adrenal cells has hindered molecular analysis of ACTH signaling pathways. Here, we ectopically expressed the mouse ACTHR in Balb/c mouse 3T3 fibroblasts to analyze ACTH signaling pathways involved in induction of fos and jun genes. Natural constitutive expression of the MC2R accessory protein (MRAP) in Balb3T3 and other mouse 3T3 fibroblasts (NIH, Swiss and 3T3-L1) renders these fibroblastic lines suitable for ectopic expression of ACTHR in its active form properly inserted into the plasma membrane at levels similar to those found in mouse Y1 adrenocortical tumor cells. The Y1 cell line is a cultured cell system well known for stably displaying normal adrenal specific metabolic pathways, ACTHR expression and ACTH functional responses. Thirty-nine sub-lines expressing ACTHR (3T3-AR transfectants) were selected for geneticin-resistance and clonally isolated after transfection of ACTHR-cDNA (in the pSVK3 mammalian plasmidial vector) into Balb3T3 fibroblasts. In addition, sixteen clonal sub-lines of Balb3T3 (3T3-0 transfectants) carrying the pSVK3 empty vector were likewise isolated. Fourteen 3T3-AR and four 3T3-0 clones were screened for response to ACTH39 in comparison with Y1 adrenocortical cells. Eight 3T3-AR clones responded to ACTH39 with activation of adenylate cyclase and induction of c-Fos protein, but the levels of, respectively, activation and induction were not strictly correlated. Other fos and jun genes were also induced by ACTH39 in 3T3-AR transfectants, which express levels of ACTHR protein similar to parental Y1 cells. Signaling pathways relevant to c-Fos induction was extensively investigated in 3 clones: 3T3-AR01 and –07 and 3T3-04. In Y1 cells, specific inhibitors (H89/PKA; PD98059/MEK; Go6983/PKC and SP600125/JNK) show that signals initiated in the ACTH/ACTHR-system activate 4 pathways to induce the c-fos gene, namely: (a) cAMP/PKA/CREB; (b) MEK/ERK1/2; (c) PKC and d) JNK1/2. In 3T3-AR transfectants, both inhibitors PD98059 and Go6983 proved completely ineffective to inhibit c-Fos induction by ACTH39, implying that MEK/ERK and PKC pathways are not involved in this process. On the other hand, SP600125 caused 85% inhibition of c-Fos induction by ACTH39 and, in addition, ACTH39 promotes JNK1/2 phosphorylation, suggesting that JNK is a major signaling pathway mediating c-Fos induction by ACTH39 in these cells. In addiction, PKA inhibitor H89 also inhibits c-Fos induction in 3T3-AR7 cells by ACTH39, implicating activation of the cAMP/PKA/CREB pathway in c-Fos induction by ACTH39. However, the cAMP derivatives db-cAMP and 8Br-cAMP, do not promote CREB phosphorylation and c-Fos induction in parental Balb3T3 and 3T3-AR transfectants, confirming previous report by others. In conclusion, expression of active ACTHR in Balb3T3 fibroblasts renders these cells responsive to ACTH with activation of cAMP/PKA/CREB and JNK pathways and, also, induction of genes from the fos and jun families. These results show that Balb 3T3-AR sublines are useful cellular systems for genetic analysis of ACTH-signaling pathways. However, activation of cAMP/PKA/CREB and JNK pathways and induction of fos and jun genes are not yet sufficient to enable ACTH for interference in morphology, migration and proliferation of Balb3T3 fibroblasts as it does in Y1 adrenocortical cells.

Sulfated mucopolysaccharides from normal Swiss 3T3 cell line and its tumorigenic mutant ST1: Possible role of chondroitin sulfates in neoplastic transformation

Abstract The sulfated mucopolysaccharide composition of normal Swiss 3T3 cell line and its tumorigenic mutant ST1 is reported. It is shown that chondroitin sulfate B and heparitin sulfate are the sulfated mucopolysaccharides of the normal 3T3 line whereas chondroitin sulfate A and heparitin sulfate are the major ones of the ST1 variant. Degradation of the chondroitin sulfates derived from both cell lines with chondroitinases B and ABC have shown that they contain only 4-sulfated disaccharides differing from each other by the type of uronic acid residue. It is also shown that the chondroitin sulfate A from the tumorigenic variant is mostly located at the cell surface whereas the chondroitin sulfate B from the normal line is less accessible to trypsinization. A relative increase of chondroitin sulfate A was also observed in 3T3 that had lost contact inhibition after successive subcultures, and in the 3T6 cell line. These combined results are in agreement with the earlier proposal that glucuronic acid-containing chondroitin sulfate plays a role in the stimulation of cell division in neoplastic and embryonic tissues.

Stimulation of heparan sulfate proteoglycan synthesis and secretion during G1 phase induced by growth factors and PMA

Fetal calf serum (FCS) and PMA (phorbol 12‐myristate‐13‐acetate) specifically stimulate the synthesis of heparan sulfate proteoglycan in endothelial cells. Staurosporine and n‐butanol, kinase inhibitors, abolish the PMA effect. Forskolin and 8‐bromo adenosine 3′:5′‐cyclic monophosphate, activators of, respectively, adenylate cyclase and protein kinase A cannot reproduce the PMA effect. The kinetics of cell entry into S phase of the endothelial cells was determined by DNA synthesis ([3H]‐thymidine and Br‐dU incorporation), and flow cytometry. The mitogenic effect of fetal calf serum is abolished by PMA. Also, PMA pre‐treatment inhibits the enhanced synthesis of heparan sulfate proteoglycan after a second PMA exposure. Remarkably, the stimulation of heparan sulfate proteoglycan synthesis by fetal calf serum and PMA seems to be mainly restricted to G1 phase. Therefore fetal calf serum and PMA cause an enhanced synthesis of heparan sulfate proteoglycan, and PMA causes a cell cycle block at G1 phase. J. Cell. Biochem. 70:563–572, 1998.

Proliferative signaling initiated in ACTH receptors

This article reviews recent results of studies aiming to elucidate modes of integrating signals initiated in ACTH receptors and FGF2 receptors, within the network system of signal transduction found in Y1 adrenocortical cells. These modes of signal integration should be central to the mechanisms underlying the regulation of the G0-->G1-->S transition in the adrenal cell cycle. FGF2 elicits a strong mitogenic response in G0/G1-arrested Y1 adrenocortical cells, that includes a) rapid and transient activation of extracellular signal-regulated kinases-mitogen-activated protein kinases (ERK-MAPK) (2 to 10 min), b) transcription activation of c-fos, c-jun and c-myc genes (10 to 30 min), c) induction of c-Fos and c-Myc proteins by 1 h and cyclin D1 protein by 5 h, and d) onset of DNA synthesis stimulation within 8 h. ACTH, itself a weak mitogen, interacts with FGF2 in a complex manner, blocking the FGF2 mitogenic response during the early and middle G1 phase, keeping ERK-MAPK activation and c-Fos and cyclin D1 induction at maximal levels, but post-transcriptionally inhibiting c-Myc expression. c-Fos and c-Jun proteins are mediators in both the strong and the weak mitogenic responses respectively triggered by FGF2 and ACTH. Induction of c-Fos and stimulation of DNA synthesis by ACTH are independent of PKA and are inhibited by the PKC inhibitor GF109203X. In addition, ACTH is a poor activator of ERK-MAPK, but c-Fos induction and DNA synthesis stimulation by ACTH are strongly inhibited by the inhibitor of MEK1 PD98059.

Oleic, Linoleic and Linolenic Acids Increase ROS Production by Fibroblasts via NADPH Oxidase Activation

The effect of oleic, linoleic and γ-linolenic acids on ROS production by 3T3 Swiss and Rat 1 fibroblasts was investigated. Using lucigenin-amplified chemiluminescence, a dose-dependent increase in extracellular superoxide levels was observed during the treatment of fibroblasts with oleic, linoleic and γ-linolenic acids. ROS production was dependent on the addition of β-NADH or NADPH to the medium. Diphenyleneiodonium inhibited the effect of oleic, linoleic and γ-linolenic acids on fibroblast superoxide release by 79%, 92% and 82%, respectively. Increased levels of p47phox phosphorylation due to fatty acid treatment were detected by Western blotting analyses of fibroblast proteins. Increased p47phox mRNA expression was observed using real-time PCR. The rank order for the fatty acid stimulation of the fibroblast oxidative burst was as follows: γ-linolenic > linoleic > oleic. In conclusion, oleic, linoleic and γ-linolenic acids stimulated ROS production via activation of the NADPH oxidase enzyme complex in fibroblasts.

Serum and hormonal regulation of the "resting-proliferative" transition in a variant of 3T3 mouse cells.

3T3 CELLS are embryonic mouse fibroblasts established in culture1. Serum depletion or “step-down” causes them to enter a “resting” state, which they leave on restoration of serum or “step-up”, entering the “proliferative” state2–4. This transition between resting (also called G0, A or R) and proliferating states seems to be the critical step in cell growth control5–7. Its study has been aided recently by several observations on extracellular growth regulators for 3T3 cells, for example the discovery of a new pituitary growth factor4,8, growth stimulation by glucocorticoids4,9 and interactions between pituitary factor and classical hormones10–12. Further studies would be greatly helped if variants or mutants of 3T3 with different growth responses towards hormones could be isolated. Here we report our finding of the variant ST1 which is inhibited by glucocorticoids, unlike 3T3 cells whose growth is stimulated by glucocorticoids4,9 (growth of fibroblasts is normally inhibited by glucocorticoids13). In this paper we discuss the value of this new cell type for studies of the resting–proliferative transition; elsewhere we shall report how the adhesion of this variant to the substrate is greatly affected by both the removal of serum and the addition of cyclic AMP.

Regulation of growth by acth in the Y-1 line of mouse adrenocortical cells

Y-1 adrenal cells were cell cycle arrested by serum starvation to characterize a G0-->G1-->S transition in these cells. Cycle arrested Y-1 cells start to enter S phase 8h after serum feeding, reaching more than 90% cells synthesizing DNA by 24h. ACTH displays a dual effect in the G0-->G1-->S transition: 2h ACTH treatment stimulates DNA synthesis initiation, but longer treatments inhibit S phase entry. This dual effect of ACTH is similar to the antagonistic actions of PMA (phorbol-12-miristate-13-acetate) on the G0-->G1-->S transition. However ACTH and PMA are likely to have different mechanisms of action. ACTH inhibitory effect requires PKA, whereas PMA inhibitory effect is not dependent on PKA. ACTH induces the proto-oncogenes c-fos and c-jun, but inhibits the expression of the c-myc proto-oncogene. PMA, on the other hand, induces equally well c-fos, c-jun and c-myc. We hypothesize that ACTH promotes G0-->G1 transition by induction of c-fos and c-jun and blocks G1-->S transition by c-myc inhibition.

Glyceraldehyde 3-Phosphate Dehydrogenase-Telomere Association Correlates with Redox Status in Trypanosoma cruzi

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a classical metabolic enzyme involved in energy production and plays a role in additional nuclear functions, including transcriptional control, recognition of misincorporated nucleotides in DNA and maintenance of telomere structure. Here, we show that the recombinant protein T. cruzi GAPDH (rTcGAPDH) binds single-stranded telomeric DNA. We demonstrate that the binding of GAPDH to telomeric DNA correlates with the balance between oxidized and reduced forms of nicotinamide adenine dinucleotides (NAD+/NADH). We observed that GAPDH-telomere association and NAD+/NADH balance changed throughout the T. cruzi life cycle. For example, in replicative epimastigote forms of T. cruzi, which show similar intracellular concentrations of NAD+ and NADH, GAPDH binds to telomeric DNA in vivo and this binding activity is inhibited by exogenous NAD+. In contrast, in the T. cruzi non-proliferative trypomastigote forms, which show higher NAD+ concentration, GAPDH was absent from telomeres. In addition, NAD+ abolishes physical interaction between recombinant GAPDH and synthetic telomere oligonucleotide in a cell free system, mimicking exogenous NAD+ that reduces GAPDH-telomere interaction in vivo. We propose that the balance in the NAD+/NADH ratio during T. cruzi life cycle homeostatically regulates GAPDH telomere association, suggesting that in trypanosomes redox status locally modulates GAPDH association with telomeric DNA.

Role of ERK/MAP kinase in mitogenic interaction between ACTH and FGF2 in mouse Y1 adrenocortical tumor cells.

In G0/G1 cell cycle-arrested Y1 adrenocortical cells FGF2 is a strong mitogen, whereas ACTH39 can be a weak mitogen or a strong anti-mitogenic agent Phosphorylated ERK1/2-MAP kinases are undetectable by Western and immunocitochemistry assay in G0/G1-arrested Y1 adrenal cells. Cell entry into S phase linearly correlates with migration of phosphorylated ERK to nucleus. FGF2 rapid and strongly triggers transient phosphorylation of ERK1/2, whereas ACTH39 is a poor ERK1/2 activator. But, the MEK1 inhibitor, PD98059 (50μM), inhibits cFos and cyclin D1 induction and DNA synthesis stimulation by both ACTH39 and FGF2, suggesting that ERK1/2 activation mediates the strong and the weak mitogenic effect of, respectively, FGF2 and ACTH39 In addition, ACTH39 antagonizes the FGF2 mitogenic effect keeping untouched ERK1/2 activation, c-Fos and cyclin D1 induction.