Retinoic acid modulation of transmembrane signaling. Analysis in F9 teratocarcinoma cells

F9 embryonal mouse teratocarcinoma cells were differentiated to a primitive endoderm-like phenotype by retinoic acid and to a parietal endoderm-like phenotype by retinoic acid in combination with dibutyryl cyclic AMP. The secretion of tissue plasminogen activator (tPA) is a characteristic of the cells displaying the differentiated phenotypes. The fundamental question of whether tPA secretion is regulated acutely by G-protein-mediated transmembrane signaling was explored. Cells differentiated to primitive and parietal endoderm demonstrated a rapid tPA response to stimulation by beta-adrenergic agonist (isoproterenol). Adenylyl cyclase activity in response to isoproterenol and GTP, but not forskolin, was greater in primitive and parietal endoderm than F9 stem cells. Both primitive and parietal endoderm cells, but not F9 stem cells, displayed beta-adrenergic stimulation of cyclic AMP accumulation. Retinoic acid induced F9 stem cells to the primitive endoderm phenotype and increased beta-adrenergic receptor levels 3-fold. Gi alpha 2 levels declined, G beta-subunits increased, and Gs alpha levels were unchanged following differentiation to primitive endoderm. In parietal endoderm cells beta-adrenergic receptors increased 2-fold over F9 stem cells, Gi alpha 2 levels declined even further than in primitive endoderm, G beta-subunits increased compared to F9 stem cells, and Gs alpha levels again were unchanged. The marked potentiation of short-term stimulation of tPA secretion in the differentiated state may be best explained by the retinoic acid-induced increase in expression of beta-adrenergic receptors coupled with a decline in Gi alpha 2 levels. Short-term regulation by G-protein-linked receptors represents a novel mode for the control of tPA secretion.     

Alterations in calcitonin and parathyroid hormone responsiveness of adenylate cyclase in F9 embryonal carcinoma cells treated with retinoic acid and dibutyryl cyclic AMP

Teratocarcinoma cells in culture offer an in vitro system for studying certain aspects of embryonic differentiation. To gain some insight into regulatory systems that might be operative during early development, we have characterized the alterations that occur in the hormonal responsiveness of the F9 embryonal carcinoma cell membrane adenylate cyclase with differentiation. Adenylate cyclase of F9 cells is stimulated in the presence of 10 μM GTP by calcitonin, prostaglandin E₁, (?) isoproterenol, and epinephrine, while parathyroid hormone is only slightly effective. Of these active hormones, calcitonin is the most potent stimulator of cyclic AMP production. Exposure of F9 cells to retinoic acid induces differentiation to parietal endodermal cells. Basal, GTP-, and fluoride-stimulated adenylate cyclase activities show a progressive increase with the retinoic acid-induced change to the endodermal phenotype. Differentiation to the endodermal cell type markedly alters the adenylate cyclase response to calcitonin and parathyroid hormone; the cyclase of endodermal cells exhibits a low response to calcitonin while parathyroid hormone dramatically enhances cyclic AMP formation. Treatment of the retinoic acid-generated endodermal cells with dibutyryl cyclic AMP converts these cells to a type exhibiting neural-like morphology. The adenylate cyclase system of these cells is only stimulated by parathyroid hormone, prostaglandin E₁, isoproterenol, and epinephrine. Calcitonin responsiveness has been lost in these cells. These variations in calcitonin and parathyroid hormone responsiveness suggest a possible regulatory role for these hormones during embryonic development. Further more, the results indicate that changes in adenylate cyclase hormonal responsiveness might serve as useful markers during early stages of differentiation.     

Plasma hyperosmolality increases G protein and 3',5'-cyclic adenosine monophosphate synthesis in the paraventricular and supraoptic nuclei

Hyperosmotic stimuli produce profound changes in cellular morphology and biosynthetic activities within the hypothalamic paraventricular and supraoptic nuclei (SON) of the rat. The mechanisms by which osmoreceptive signals are transduced within these nuclei are poorly understood. We examined several components of the cAMP-associated second messenger system after giving rats 2% saline to drink for one week, a strong hyperosmotic stimulus. We found that mRNA levels for both the stimulatory and inhibitory guanine-nucleotide binding protein alpha-subunits were increased in the paraventricular nucleus and SON. In the SON, these changes were accompanied by increased basal cAMP levels, cholera toxin-stimulated adenylate cyclase, and Gs alpha. Our results suggest that Gs alpha levels are not saturated with respect to adenylate cyclase coupling and that osmoreception activates the cAMP second messenger system.     

Agonist regulation of beta-adrenergic receptor mRNA. Analysis in S49 mouse lymphoma mutants

Agonist-promoted down-regulation of beta-adrenergic receptor mRNA was investigated in S49 mouse lymphoma variants with mutations in elements of hormone-sensitive adenylate cyclase. In wild-type cells steady-state levels of beta-adrenergic receptor mRNA were established by DNA-excess solution hybridization to be 1.72 +/- 0.08 (n = 8) amol/microgram total cellular RNA. Receptor mRNA levels declined 35-45% in response to stimulation by the beta-adrenergic agonist (-)isoproterenol or forskolin as described previously in DDT1 MF-2 cells (Hadcock, J. R., and Malbon, C. C. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 5021-5025). Agonist-promoted cAMP accumulation and down-regulation of receptor mRNA were analyzed in three variants with mutations in Gs alpha (H21a, unc, cyc-) and a single variant lacking cAMP-dependent protein kinase activity (kin-). H21a (Gs alpha coupled to receptor, but not to adenylate cyclase), unc (Gs alpha uncoupled from receptor), and cyc- (lacking Gs alpha) variants accumulated cAMP and down-regulated beta AR mRNA in response to forskolin. In unc and cyc- cells isoproterenol failed to stimulate cAMP; accumulation and down-regulation of receptor mRNA was not observed. H21a cells, in contrast, displayed agonist-promoted regulation of beta-adrenergic receptor mRNA but only basal levels of cAMP accumulation in response to isoproterenol. The kin- cells displayed cAMP accumulation in response to forskolin as well as to isoproterenol but no down-regulation of receptor mRNA or receptor expression. Taken together these data demonstrate several features of agonist-promoted down-regulation of mRNA: (i) cAMP-dependent protein kinase activity is required for down-regulation of mRNA (kin-), although elevated cAMP accumulation is not (H21a); (ii) functional receptor-Gs coupling is required (H21a), and clones lacking Gs alpha (cyc-) or receptor Gs coupling (unc) lack the capacity to down-regulate mRNA in response to agonist; and (iii) in the presence of basal levels of cAMP and cAMP-dependent protein kinase activity, functional receptor-Gs coupling (H21a) to some other effector other than adenylate cyclase may be propagating the signal.     

Retinoic acid-induced blr1 expression promotes ERK2 activation and cell differentiation in HL-60 cells

Retinoids are known to induce the differentiation and cell cycle arrest of human myeloid leukemia cells in vitro. Differential display was used to identify putative early regulatory genes that are differentially expressed in HL-60 human promyelocytic leukemia cells treated with retinoic acid. One of the cDNAs cloned encodes sequences identifying Burkitt's lymphoma receptor 1 (BLR1), a recently described chemokine receptor. Northern blot analysis demonstrates that blr1 mRNA expression increases within 9 h of retinoic acid treatment, well before functional differentiation or G(1)/G(0) growth arrest at 48 h or onset of morphological changes, suggesting a possible regulatory function. The expression of blr1 mRNA is transient, peaking at 72 h when cells are differentiated. blr1 mRNA also is induced by other differentiation-inducing agents, 1alpha,25-dihydroxyvitamin D(3) and DMSO. Induction of blr1 mRNA by retinoic acid is not blocked by the protein synthesis inhibitor cycloheximide. In HL-60 cells stably transfected with blr1 cDNA, ectopic expression of blr1 causes an increase in ERK2 MAPK activation and promotes retinoic acid-induced G(1)/G(0) growth arrest and cell differentiation. The early expression of blr1 mRNA during differentiation, its ability to increase ERK2 activation, and its enhancement of retinoic acid-induced differentiation suggest that blr1 expression may be involved in retinoic acid-induced HL-60 differentiation.     

Prolonged activation of inhibitory somatostatin receptors increases adenylate cyclase activity in wild-type and Gs alpha-deficient (cyc-) S49 mouse lymphoma cells.

Many cells develop enhanced adenylate cyclase activity after prolonged exposure to drugs that acutely inhibit the enzyme and it has been suggested that this adaptation may be due to an increase in Gs alpha. We have treated wild-type and Gs alpha-deficient cyc- S49 mouse lymphoma cells with a stable analogue (SMS 201-995) of the inhibitory agonist somatostatin. After incubation with SMS for 24 h, the forskolin-stimulated cAMP synthetic rate in intact cyc- cells was increased by 76%, similar to the increase found in the wild-type cells. Forskolin-stimulated adenylate cyclase activity in the presence of Mn2+ was also increased in membranes prepared from SMS-treated cyc- cells; however, guanine nucleotide-mediated inhibition of adenylate cyclase activity was not changed despite a small decrease in inhibitory Gi alpha subunits detected by immunoblotting. Pretreatment of cyc- cells with pertussis toxin prevented SMS from inducing the enhancement of forskolin-stimulated cAMP accumulation in intact cells. After chronic incubation of cyc- cells with SMS, exposure to N-ethylmaleimide, which abolished receptor-mediated inhibition of cAMP accumulation, did not attenuate the enhanced rate of forskolin-stimulated cAMP synthesis compared to N-ethylmaleimide-treated controls. These results with cyc- cells demonstrate that an adaptive increase in adenylate cyclase activity induced by chronic treatment with an inhibitory drug can occur in the absence of expression of Gs alpha.     

Thyroid hormones regulate G-protein beta-subunit mRNA expression in vivo

Thyroid hormones exert "permissive effects" on the hormone-sensitive adenylate cyclase. Regulation of the expression of Gi (Gi alpha 2) and Gs by thyroid hormones in vivo was investigated at the level of mRNA. Steady-state levels of the mRNA for Gi alpha 2 and Gs alpha, as well as the G beta-subunits, were quantified using DNA excess solution hybridization analysis. Regulation of protein and mRNA expression in adipose tissue was investigated in hypothyroid, euthyroid, and hyperthyroid rats. In euthyroid animals, steady-state levels of mRNA (amol/microgram RNA) were 13.8, 5.9, and 5.7 for Gs alpha, Gi alpha 2, and G beta 1,2, respectively. Activation of adenylate cyclase by Gs is unaffected by thyroid status. Both Gs alpha and Gs alpha mRNA levels in hypothyroid rats were the same as those of controls (euthyroid). The inhibitory control of adenylate cyclase, in contrast, is markedly potentiated in hypothyroid rats. The expression of G1 alpha s and G beta-subunits was increased in hypothyroidism. Whereas Gi alpha 2 mRNA levels remained essentially unchanged, G beta 1,2 mRNA levels were observed to increase 45% in the hypothyroid state. In the hyperthyroid state G beta 1,2 mRNA levels were observed to decline by 35%. Regulation of G-protein subunit expression, at the level of mRNA, appears to be one component of permissive hormone action on transmembrane signalling.     

Cross-regulation between G-protein-mediated pathways. Stimulation of adenylyl cyclase increases expression of the inhibitory G-protein, Gi alpha 2

The hormone-sensitive adenylyl cyclase system is under dual control, receiving both stimulatory and inhibitory inputs. Guanine nucleotide-binding regulatory proteins (G-proteins) transduce signals from cell surface receptors to effectors such as adenylyl cyclase. Hormonal stimulation is propagated via Gs, inhibition by Gi. Persistent (24-h) activation of the stimulatory pathway of adenylyl cyclase by the diterpene forskolin or the beta-adrenergic agonist isoproterenol in S49 mouse lymphoma cells enhanced the effects of somatostatin mediated via the inhibitory pathway of adenylyl cyclase. Stimulating cells with forskolin or isoproterenol for 24 h resulted in a 3-fold increase in the steady-state levels of Gi alpha 2 and a 25% decline in Gs alpha, as quantified by immunoblotting. Within 12 h of stimulation of adenylyl cyclase, Gi alpha 2 mRNA levels increased 4-fold, measured by DNA-excess solution hybridization. Gs alpha mRNA levels, in contrast, increased initially (25%), but then declined to 75% of control. In S49 variants that lack functional protein kinase A (kin-), stimulation by isoproterenol failed to alter Gi alpha 2 expression at either the protein or the mRNA levels. A 3-fold increase in relative synthesis rate and no change in the half-life (approximately 80 h) of Gi alpha 2 was observed in response to forskolin stimulation. Although Gs alpha synthesis increased (70%) modestly in response to forskolin stimulation, the half-life of Gs alpha actually decreased from 55 h in naive cells to 34 h in treated cells. Thus, the two G-protein-mediated pathways controlling adenylyl cyclase display "cross-regulation." Persistent activation of the stimulatory pathway increases Gi alpha 2 mRNA and expression. Transiently elevated Gs alpha mRNA levels are counterbalanced by a reduction in the half-life of the protein.     

Gs alpha mediates epidermal growth factor-elicited stimulation of rat cardiac adenylate cyclase

In an earlier study we demonstrated that epidermal growth factor (EGF) increases the cellular accumulation of cAMP in perfused rat hearts by stimulating the cardiac adenylate cyclase via a stimulatory GTP-binding protein (Nair, B. G., Rashed, H. M., and Patel, T. B. (1989) Biochem. J. 264, 563-571). Employing antiserum, CS1, generated against a synthetic decapeptide RMHLRQYELL representing the carboxyl terminus of Gs alpha, the involvement of Gs in mediating the effects of EGF on cardiac adenylate cyclase was further investigated. The CS1 antiserum specifically recognized two forms, (52 and 40 kDa) of Gs alpha in rat cardiac membranes; the 52 kDa being the predominant species. In functional assays of adenylate cyclase activity, the CS1 antiserum did not alter either aluminum fluoride- or forskolin-stimulated adenylate cyclase activity. Similarly, basal adenylate cyclase activity in the absence of guanyl-5'-yl imidodiphosphate (Gpp(NH)p) was also not altered by the CS1 antiserum. However, as compared with controls performed in the presence of non-immune serum, preincubation of cardiac membranes with the CS1 antiserum resulted in a concentration-dependent inhibition of Gpp(NH)p-, isoproterenol-, and EGF-stimulated activities. In experiments which monitored Gi function as the ability of different G(pp)NHp, (-)N6-(R-phenylisopropyl)adenosine and carbachol to inhibit forskolin-stimulated adenylate cyclase, CS1 antiserum by inhibiting Gs, increased the apparent activity of Gi. Overall, our data demonstrate that the CS1 antiserum can specifically inhibit Gs function and therefore the stimulation of adenylate cyclase by agonists whose actions are mediated by Gs. In this respect, the data presented here demonstrate that Gs is the G-protein involved in mediating EGF-elicited stimulation of cardiac adenylate cyclase. Additionally, the finding that CS1 antiserum can overcome the effects of Gpp(NH)p on Gs, but not Gi, suggests that the carboxyl-terminal region of Gs alpha is important in the interactions with GTP or its analogs.     

Retinoic acid abolishes the calcitonin gene-related peptide autocrine system in F9 teratocarcinoma cells

Calcitonin gene-related peptide (CGRP), expressed predominantly in F9 embryonal carcinoma cells, is both a potent chemotactic agent and an autocrine growth factor for these cells. We analyzed the effect of retinoic acid (RA)-induced differentiation of F9 cells into primitive parietal endoderm-like cells, on CGRP production and the CGRP responsiveness of these cells. Poly(A) RNA extracted from F9 cells and analysed by Northern blotting and hybridization with a CGRP probe showed a specific band of about 1200 bases corresponding to mature CGRP mRNA. This band was not detected in F9 cells treated for 6 days with RA (differentiated primitive parietal endoderm-like cells) or in PYS cells (established parietal endoderm-like cell line). During RA-induced differentiation of F9 cells, CGRP mRNA levels fell within 24 h after treatment and were almost undetectable after 2 days. RA treatment also reduced CGRP secretion by F9 cells; the effect was maximal at 3 days and remained stable thereafter. Similarly, RA rapidly reduced adenylate cyclase responsiveness to chicken CGRP (cCGRP) and human CGRP (hCGRP). An 80% fall in cAMP release into the culture medium in the presence of CGRP was observed after 24 h of RA treatment. These results demonstrate that RA rapidly abolishes the CGRP autocrine system involved in the proliferation of F9 cells, at the same time inducing their differentiation into primitive parietal endoderm. They point to the interaction between retinoic acid and growth factors in the regulation of cell proliferation and differentiation. J. Cell. Biochem. 64:447–457. © 1997 Wiley-Liss, Inc.     

Immunoprecipitation of adenylate cyclase with an antibody to a carboxyl-terminal peptide from Gs alpha

An antibody (RM) raised against the carboxyl-terminal decapeptide of the alpha subunit of the stimulatory guanine nucleotide regulatory protein (Gs alpha) has been used to study the interaction of Gs alpha with bovine brain adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1]. RM antibody immunoprecipitated about 60% of the solubilized adenylate cyclase preactivated with either GTP-gamma-S or AlF4-. In contrast, RM antibody immunoprecipitated about 5% of the adenylate cyclase not preactivated (control) and 15% of the adenylate cyclase pretreated with forskolin. Adenylate cyclase solubilized from control membranes or GTP-gamma-S preactivated membranes was partially purified by using forskolin-agarose affinity chromatography. The amount of Gs alpha protein in the partially purified preparations was determined by immunoblotting with RM antibody. There was 3-fold more Gs alpha detected in partially purified adenylate cyclase from preactivated membranes than in the partially purified adenylate cyclase from control membranes. Partially purified adenylate cyclase from preactivated membranes was immunoprecipitated with RM antibody and the amount of adenylate cyclase activity immunoprecipitated (65% of total) corresponded to the amount of Gs alpha protein immunoprecipitated. Only 15% of the partially purified adenylate cyclase from control membranes was immunoprecipitated. The presence of other G proteins in the partially purified preparations of adenylate cyclase was investigated by using specific antisera that detect Go alpha, Gi alpha, and G beta. The G beta protein was the only subunit detected in the partially purified preparations of adenylate cyclase and the amount of G beta was about the same in adenylate cyclase from preactivated membranes and from control membranes.(ABSTRACT TRUNCATED AT 250 WORDS)     

MicroRNA-181a-mediated downregulation of AC9 protein decreases intracellular cAMP level and inhibits ATRA-induced APL cell differentiation

AC9 is one of the adenylate cyclase (AC) isoforms, which catalyze the conversion of ATP to cAMP, an important second messenger. We previously found that the integration of cAMP/PKA pathway with nuclear receptor-mediated signaling was required during all-trans retinoic acid (ATRA)-induced maturation of acute promyelocytic leukemia (APL) cells. Here we showed that AC9 could affect intracellular cAMP level and enhance the trans-activity of retinoic acid receptor. Knockdown of AC9 in APL cell line NB4 could obviously inhibit ATRA-induced differentiation. We also demonstrated that miR-181a could decrease AC9 expression by targeting 3′UTR of AC9 mRNA, finally controlling the production of intracellular cAMP. The expression of miR-181a itself could be inhibited by CEBPα, probably accounting for the differential expression of miR-181a in NB4 and ATRA-resistant NB4-R1 cells. Moreover, we found that AC9 expression was relatively lower in newly diagnosed or relapsed APL patients than in both complete remission and non-leukemia cases, closely correlating with the leukemogenesis of APL. Taken together, our studies revealed for the first time the importance of miR-181a-mediated AC9 downregulation in APL. We also suggested the potential value of AC9 as a biomarker in the clinical diagnosis and treatment of leukemia.     

Neuroblastoma Differentiation Involves the Expression of Two Isoforms of the α-Subunit of Go

The regulation of GTP-binding proteins (G proteins) was examined during the course of differentiation of neuroblastoma N1E-115 cells. N1E-115 cell membranes possess three Bordetella pertussis toxin (PTX) substrates assigned to alpha-subunits (G alpha) of Go (a G protein of unknown function) and "Gi (a G protein inhibitory to adenylate cyclase)-like" proteins and one substrate of Vibrio cholerae toxin corresponding to an alpha-subunit of Gs (a G protein stimulatory to adenylate cyclase). In undifferentiated cells, only one form of Go alpha was found, having a pI of 5.8 Go alpha content increased by approximately twofold from the undifferentiated state to 96 h of cell differentiation. This is mainly due to the appearance of another Go alpha form having a pI of 5.55. Both Go alpha isoforms have similar sizes on sodium dodecyl sulfate-polyacrylamide gels, are recognized by polyclonal antibodies to bovine brain Go alpha, are ADP-ribosylated by PTX, and are covalently myristylated in whole N1E-115 cells. In addition, immunofluorescent staining of N1E-115 cells with Go alpha antibodies revealed that association of Go alpha with the plasma membrane appears to coincide with the expression of the most acidic isoform and morphological cell differentiation. In contrast, the levels of both Gi alpha and Gs alpha did not significantly change, whereas that of the common beta-subunit increased by approximately 30% over the same period. These results demonstrate specific regulation of the expression of Go alpha during neuronal differentiation.     

Disabled-2 mediates c-Fos suppression and the cell growth regulatory activity of retinoic acid in embryonic carcinoma cells

F9 embryonic stem cell-like teratocarcinoma cells are widely used to study early embryonic development and cell differentiation. The cells can be induced by retinoic acid to undergo endodermal differentiation. The retinoic acid-induced differentiation accompanies cell growth suppression, and thus, F9 cells are also often used as a model for analysis of retinoic acid biological activity. We have recently shown that MAPK activation and c-Fos expression are uncoupled in F9 cells upon retinoic acid-induced endodermal differentiation. The expression of the candidate tumor suppressor Disabled-2 is induced and correlates with cell growth suppression in F9 cells. We were not able to establish stable Disabled-2 expression by cDNA transfection in F9 cells without induction of spontaneous cell differentiation. Transient transfection of Dab2 by adenoviral vector nevertheless suppresses Elk-1 phosphorylation, c-Fos expression, and cell growth. In PA-1, another teratocarcinoma cell line of human origin that has no or very low levels of Disabled-2, retinoic acid fails to induce Disabled-2, correlating with a lack of growth suppression, although PA-1 is responsive to retinoic acid in morphological change. Transfection and expression of Disabled-2 in PA-1 cells mimic the effects of retinoic acid on growth suppression; the Disabled-2-expressing cells reach a much lower saturation density, and serum-stimulated c-Fos expression is greatly suppressed and disassociated from MAPK activation. Thus, Dab2 is one of the principal genes induced by retinoic acid involved in cell growth suppression, and expression of Dab2 alone is sufficient for uncoupling of MAPK activation and c-Fos expression. Resistance to retinoic acid regulation in PA-1 cells likely results from defects in retinoic acid up-regulation of Dab2 expression.     

Cellular HSP90 (HSP86) mRNA level and in vitro differentiation of mouse embryonal carcinoma (F9) cells

Treatment of mouse embryonal carcinoma (F9) cells with retinoic acid, an inducer of F9 cell differentiation, greatly increased the level of mRNA specific to one of the heat-shock proteins (HSP86). Experiments including the one employing differentiation-resistant mutant F9 cells suggested that the increase represents early molecular events associated with the embryonal differentiation. The increased HSP86 mRNA declined to the original level during further incubation. The presence of cyclic AMP, which stimulates conversion of the retinoic acid-induced primitive endoderm cells to parietal endoderm cells, prevented the decline. These results suggest that not only the elevation of HSP86 mRNA level represents early molecular events in F9 cell differentiation but also that sustaining the elevated level (by cyclic AMP) is associated with further differentiation of the embryonal cells.     

Induction of expression of the alpha v beta 1 and alpha v beta 3 integrin heterodimers during retinoic acid-induced neuronal differentiation of murine embryonal carcinoma cells

All-trans-retinoic acid, an endogenous morphogen, induced neuronal differentiation of P19 murine embryonal carcinoma cells. Peak differentiation, as judged by the elaboration of neuronal processes, occurred 8 days after exposure of the cells to 0.5 mM retinoic acid, a concentration known to induce neuronal differentiation. An examination of the expression of the extracellular matrix receptors, integrins, during this retinoic acid-induced differentiation period, demonstrated a specific and strong induction of expression of two polypeptides (130 and 115 kDa) immunoprecipitated with an anti-human vitronectin receptor antiserum. The expression of a 90-kDa polypeptide, also immunoprecipitating with this antiserum was induced as well, but to a much smaller extent. The expression of a 96-kDa polypeptide immunoprecipitated by this antiserum and present in the untreated cells was not induced by retinoic acid. The increase in the expression of these polypeptides paralleled the neuronal differentiation of the P19 embryonal carcinoma cells. The expression of these integrins was not induced in a variant of the P19 cells, P19RAC65, which are resistant to differentiation induction by retinoic acid. Utilizing integrin subunit-specific anti-cytoplasmic peptide antibodies together with immunoprecipitation and Western blot analysis, the 130- and 115-kDa polypeptides were identified as the integrin alpha v and beta 1 subunits, respectively. The 90-kDa polypeptide, also induced by retinoic acid, was identified as beta 3, whereas the identity of the uninduced 96-kDa polypeptide remains unclear as yet. Peptide map analysis of deglycosylated polypeptides demonstrated that the 90- and 96-kDa polypeptides are distinct proteins and that the 115-kDa polypeptides immunoprecipitated with either anti-alpha v or anti-beta 1 antibodies are identical, further establishing that the 115-kDa polypeptide associating with alpha v is beta 1. The retinoic acid-induced expression of beta 1 occurred at the level of mRNA expression which also paralleled neuronal differentiation, but peaked slightly ahead of the cell surface expression of beta 1. The expression of other beta 1-associated alpha subunits was not induced by retinoic acid in these cells. These data demonstrate that retinoic acid strongly induces the expression of the integrin heterodimer alpha v beta 1 and also, to a smaller extent, the expression of alpha v beta 3. The retinoic acid-induced, high level surface expression of the alpha v beta 1 heterodimer is tightly correlated with the induction of neuronal differentiation by retinoic acid. This finding suggests an important role for the alpha v beta 1 heterodimer in the neuronal differentiation process.     

Increases in mRNA concentrations of the alpha and beta subunits of prolyl 4-hydroxylase accompany increased gene expression of type IV collagen during differentiation of mouse F9 cells.

Mouse F9 teratocarcinoma stem cells differentiate in monolayer cultures in the presence of retinoic acid, dibutyryl cAMP, and isobutyl methylxanthine. This differentiation is associated with a marked increase in the synthesis rates and mRNA concentrations of basement membrane proteins such as type IV collagen. We report here that the differentiation also involves an increase of up to 50-fold in the concentrations of the mRNAs for the alpha and beta subunits of prolyl 4-hydroxylase, the enzyme required for the cotranslational and post-translational hydroxylation of proline residues in collagens. The time courses and magnitudes of increases in these two mRNA concentrations were similar to those observed in the same experiments for the mRNA of the alpha chain of type IV collagen. In the differentiated F9 cells the concentration of the alpha subunit mRNA was about 30% of the beta subunit mRNA concentration. Northern blot analyses indicated that the sizes of the alpha and beta subunit mRNAs in the differentiated mouse F9 cells are similar to those in human skin fibroblasts. The F9 cell differentiation system appears to provide a useful model for studies on the regulation of prolyl 4-hydroxylase synthesis.     

Altered activation by calcitonin and inhibition by somatostatin of human embryonal carcinoma cell adenylate cyclase with retinoic acid-induced differentiation

Human teratocarcinoma cells in culture offer an in vitro system for studying certain aspects of embryonic differentiation. To gain some insight into regulatory systems that might be operative during early human development, we have characterized the alterations that occur in the hormonal responsiveness of human embryonal carcinoma cell adenylate cyclase with differentiation in response to 10 microM retinoic acid. Two cell lines CL12 and CL13, cloned from Tera 2 cells by Dr. C. F. Graham, have been used in these studies. Adenylate cyclase of CL12 and CL13 cells is stimulated in the presence of 10 microM GTP by epinephrine and calcitonin, with calcitonin being the most potent stimulator of cyclic AMP production. Exposure of these cells to retinoic acid leads to an arrest in growth and within 6 days to a differentiated cell population with a stable nonreversible phenotype. No changes in basal, GTP- and fluoride-stimulated adenylate cyclase activities are observed with retinoic acid treatment, but the cyclase of differentiated cells exhibits a greater stimulation by calcitonin (7.5-fold) and the appearance of a somatostatin inhibitory effect. Somatostatin specifically inhibits, by 25%, the hormonal stimulation of adenylate cyclase of cells treated for 5 days with retinoic acid. The increase in calcitonin stimulation of adenylate cyclase activity of the differentiated cells is related to an increase (congruent to 3-fold) in the number of hormonal receptors and not to a significant change in receptor binding affinity (Kd 4.6 X 10(8) M-1). These alterations in calcitonin and somatostatin responsiveness suggest a possible regulatory role for these hormones during embryonic development. Furthermore, the results indicate that changes in adenylate cyclase hormonal responsiveness might serve as useful markers during early stages of human embryonal carcinoma cell differentiation.