Overlapping functions of the pRb family in the regulation of rRNA synthesis.

The "pocket" proteins pRb, p107, and p130 are a family of negative growth regulators. Previous studies have demonstrated that overexpression of pRb can repress transcription by RNA polymerase (Pol) I. To assess whether pRb performs this role under physiological conditions, we have examined pre-rRNA levels in cells from mice lacking either pRb alone or combinations of the three pocket proteins. Pol I transcription was unaffected in pRb-knockout fibroblasts, but specific disruption of the entire pRb family deregulated rRNA synthesis. Further analysis showed that p130 shares with pRb the ability to repress Pol I transcription, whereas p107 is ineffective in this system. Production of rRNA is abnormally elevated in Rb(-/-) p130(-/-) fibroblasts. Furthermore, overexpression of p130 can inhibit an rRNA promoter both in vitro and in vivo. This reflects an ability of p130 to bind and inactivate the upstream binding factor, UBF. The data imply that rRNA synthesis in living cells is subject to redundant control by endogenous pRb and p130.

Regulation of RNA polymerase III transcription during cell cycle entry.

Increased rates of RNA polymerase (pol) III transcription constitute a central feature of the mitogenic response, but little is known about the mechanism(s) responsible. We demonstrate that the retinoblastoma protein RB plays a major role in suppressing pol III transcription in growth-arrested fibroblasts. RB knockout cells are compromised in their ability to down-regulate pol III following serum withdrawal. RB binds and represses the pol III-specific transcription factor TFIIIB during G(0) and early G(1), but this interaction decreases as cells approach S phase. Full induction of pol III coincides with mid- to late G(1) phase, when RB becomes phosphorylated by cyclin D- and E-dependent kinases. TFIIIB only associates with the underphosphorylated form of RB, and overexpression of cyclins D and E stimulates pol III transcription in vivo. The RB-related protein p130 also contributes to the repression of TFIIIB in growth-arrested fibroblasts. These observations provide insight into the mechanisms responsible for controlling pol III transcription during the switch between growth and quiescence.

RNA polymerase III transcription factor IIIB is a target for repression by pocket proteins p107 and p130.

RNA polymerase III (Pol III) transcription is subject to repression by the retinoblastoma protein RB, both in vitro and in vivo (R. J. White, D. Trouche, K. Martin, S. P. Jackson, and T. Kouzarides, Nature 382:88-90, 1996). This is achieved through a direct interaction between RB and TFIIIB, a multisubunit factor that is required for the expression of all Pol III templates (C. G. C. Larminie, C. A. Cairns, R. Mital, K. Martin, T. Kouzarides, S. P. Jackson, and R. J. White, EMBO J. 16:2061-2071, 1997; W.-M. Chu, Z. Wang, R. G. Roeder, and C. W. Schmid, J. Biol. Chem. 272:14755-14761, 1997). p107 and p130 are two closely related proteins that display 30 to 35% identity with the RB polypeptide and share some of its functions. We show that p107 and p130 can both repress Pol III transcription in transient transfection assays or when added to cell extracts. Pull-down assays and immunoprecipitations using recombinant components demonstrate that a subunit of TFIIIB interacts physically with p107 and p130. In addition, endogenous TFIIIB is shown by cofractionation and coimmunoprecipitation to associate stably with both p107 and p130. Disruption of this interaction in vivo by using the E7 oncoprotein of human papillomavirus results in a marked increase in Pol III transcription. Pol III activity is also deregulated in fibroblasts derived from p107 p130 double knockout mice. We conclude that TFIIIB is targeted for repression not only by RB but also by its relatives p107 and p130.

Transcription by RNA polymerases I and III: a potential link between cell growth, protein synthesis and the retinoblastoma protein.

The rate of protein synthesis is a critical determinant of cellular growth. Abnormal activation of this process is a frequent feature of transformed and tumour cells. Several distinct components of the translation apparatus have been shown to be deregulated in response to malignant transformation. Indeed, overexpression of certain translation factors has been found to predispose cells to transformation or even initiate it. The latest twist to this story comes from the discovery that the retinoblastoma protein RB plays a major role in restricting the production of tRNA and rRNA. RB is an important tumour suppressor. Its ability to limit the synthesis of these principle determinants of biosynthetic capacity could provide a mechanism for restraining cell growth. The loss of this control may constitute a significant step towards tumour progression.

Cyclic AMP inhibitors inhibits PDGF-stimulated mitogen-activated protein kinase activity in rat aortic smooth muscle cells via inactivation of c-Raf-1 kinase and induction of MAP kinase phosphatase-1.

In rat aortic smooth muscle cells (RASMC), pretreatment with forskolin inhibited the activation of p42/44 isoforms of mitogen-activated protein kinase (MAP) kinase stimulated in response to low concentrations of PDGF (10 ng/ml). This correlated with a strong inhibition of PDGF-stimulated MEK and C-Raf-1 kinase activity. However, the effect of forskolin could be surmounted by increasing the concentration of PDGF. Under such conditions forskolin was only effective against prolonged MAP kinase activation. The ability of forskolin to inhibit the late phase of MAP kinase activity was reversed by pretreatment of the cells with cycloheximide, suggesting the involvement of a protein synthesis step. This was not due to effects upstream of MAP kinase since PDGF-stimulated MEK activation was decreased by cycloheximide, an effect potentiated by forskolin. Forskolin stimulated the induction of the dual specific phosphatase MAP kinase phosphatase-1 (MKP-1), although this effect was small relative to levels induced by PDGF and angiotensin II. However, PDGF stimulated induction of MKP-1 was abolished by the protein kinase A inhibitor H89 and this correlated with the reversal of forskolin-mediated inhibition of PDGF-stimulated MAP kinase activity. These studies implicate a role for intracellular cyclic AMP in at least two aspects of MAP kinase signaling, including both the inhibition of Raf-1 activation and the induction of MKP-1.

Role of receptor desensitization, phosphatase induction and intracellular cyclic AMP in the termination of mitogen-activated protein kinase activity in UTP-stimulated EAhy 926 endothelial cells.

We have investigated the mechanisms that bring about the termination of mitogen-activated protein kinase (MAP kinase) activation in response to UTP in EAhy 926 endothelial cells. UTP-stimulated MAP kinase activity was transient, returning to basal values by 60 min. At this time MAP kinase activation was desensitized; re-application of UTP did not further activate MAP kinase, full re-activation of MAP kinase being only apparent after a 1-2 h wash period. However, activation of MAP kinase by UTP could be sustained beyond 60 min by preincubation of the cells with the protein synthesis inhibitor cycloheximide. UTP also stimulated expression of MAP kinase phosphatase-1 and this was abolished after pretreatment with cycloheximide. Pretreatment of cells with forskolin abolished the initial activation of MAP kinase kinase or c-Raf-1 by UTP, but only affected MAP kinase activity during prolonged stimulation. The effect of forskolin on prolonged MAP kinase activation was also prevented by cycloheximide. These results suggest that the termination of MAP kinase activity in response to UTP involves a number of interacting mechanisms including receptor desensitization and the induction of a phosphatase. However, several pieces of evidence do not support a major role for MAP kinase phosphatase-1 in termination of the MAP kinase signal. Raising intracellular cyclic AMP may also be involved but only after an initial protein-synthesis step and by a mechanism that does not involve the inactivation of c-Raf-1 or MAP kinase kinase.

Stimulation by the nucleotides, ATP and UTP of mitogen-activated protein kinase in EAhy 926 endothelial cells.

1. We have investigated the characteristics of activation of the 42kDa isoform of mitogen-activated protein (MAP) kinase in response to various nucleotides in the endothelial cell line EAhy 926. 2. Adenosine 5'-triphosphate (ATP) in the concentration range 0.1-100 microM stimulated the rapid and transient tyrosine phosphorylation and activation of the 42 kDa isoform of MAP kinase in EAhy 926 endothelial cells which peaked at 2 min and returned to basal values by 60 min. ATP also stimulated a similar response in primary cultured bovine aortic endothelial cells. 3. Uridine 5' triphosphate (UTP) also stimulated the 42 kDa isoform of MAP kinase with similar potency to ATP (EC50 values 5.1 +/- 0.2 microM for UTP; 2.9 +/- 0.8 microM for ATP), whilst the selective P2Y-purinoceptor agonist, 2-methylthioATP (2-meSATP) was without effect up to concentrations of 100 microM. In bovine aortic endothelial cells however, UTP and 2-meSATP both stimulated MAP kinase. 4. Pretreatment of cells for 24 h with 12-O tetradecanoyl phorbol 13-acetate resulted in the loss of the alpha and epsilon isoforms of protein kinase C (PKC) and virtual abolition of nucleotide-stimulated MAP kinase activity (> 90% inhibition). 5. Preincubation for 30 min with the PKC inhibitor, Ro-31 8220 (10 microM) reduced MAP-kinase activation at 2 min but potentiated the response at 60 min. 6. Removal of extracellular calcium in the presence of EGTA reduced the MAP kinase activation in response to UTP by approximately 30-50%. 7. Pretreatment with pertussis toxin (18 h, 50 ng ml-1) did not significantly affect the UTP-mediated activation of pp42 MAP kinase. 8. These results show that in the EAhy 926 endothelial cell line, nucleotides stimulate activation of MAP kinase in a protein kinase C-dependent manner through interaction with a P2U-purinoceptor.

The role of protein kinase C in activation and termination of mitogen-activated protein kinase activity in angiotensin II-stimulated rat aortic smooth-muscle cells.

Mitogen-activated protein (MAP) kinases are a family of serine/threonine kinases activated by both tyrosine kinase and G-protein-linked receptor agonists. In rat aorta vascular smooth-muscle cells (VSMC), vasoconstrictors, angiotension II (AII), and alpha-thrombin (alpha-thr), as well as platelet-derived growth factor beta beta (PDGF) stimulated the tyrosine phosphorylation and activation of MAP kinase in a time- and concentration-dependent manner. Pre-treatment of cells with the protein kinase C (PKC) inhibitor Ro-318220, inhibited the initial increase in tyrosine phosphorylation of MAP kinase in response to vasoconstrictors, suggesting the involvement of PKC. Four isoforms of PKC were identified in VSMC by western blotting: alpha, beta, epsilon, and zeta. Downregulation of PKC alpha and PKC epsilon isoforms following chronic phorbol myristate 12, 13-acetate (PMA) pre-treatment resulted in the abolition of AII-stimulated MAP kinase activation. Selective downregulation of PKC alpha following pre-treatment with bryostatin 1 did not affect AII-stimulated MAP kinase. Preincubation of cells with Ro-318220 enhanced the activation of MAP kinase at later time points. In addition, Ro-318220 pre-treatment inhibited the induction by AII of a novel transcriptionally regulated phosphatase, MAP kinase phosphatase-1 (MKP-1). However, AII-mediated activation of MAP kinase was not prolonged by cycloheximide pre-treatment and was not maintained indefinitely by Ro-318220. These results demonstrate a specific role for the Ca(2+)-independent PKC isoform, PKC epsilon, in the activation of MAP kinase in response to vasoconstrictors, and suggest that PKC-mediated induction of MKP-1 plays no role in the termination of transiently activated MAP kinase.

Regulation of lysophosphatidic acid-stimulated tyrosine phosphorylation of mitogen-activated protein kinase by protein kinase C- and pertussis toxin-dependent pathways in the endothelial cell line EAhy 926.

In the endothelial cell line EAhy 926, 1-oleoyl-lysophosphatidic acid (LPA) stimulated the tyrosine phosphorylation of the pp42 isoform of mitogen-activated protein (MAP) kinase. Maximum phosphorylation was observed within 5 min of LPA addition, but the response was sustained for up to 120 min. Re-addition of LPA after 60 min stimulated a further sustained increase in the tyrosine phosphorylation of MAP kinase. In cells pretreated with phorbol 12-myristate 13-acetate (PMA; 24 h) or preincubated with the protein kinase C inhibitor Ro-318220, LPA-induced tyrosine phosphorylation of pp42 MAP kinase was substantially reduced at 2 min but potentiated at 60 min. Ro-318220 in combination with either PMA or pertussis toxin pretreatment abolished the LPA response at all time points, suggesting an involvement of protein kinase C in the pertussis toxin-sensitive part of the pathway. Agents which raised intracellular cyclic AMP levels did not affect the initial phase of LPA-stimulated MAP kinase activation, but abolished the late phase. However, this effect was prevented by Ro-318220, implicating a greater role for protein kinase C than protein kinase A in the regulation of sustained MAP kinase responses. LPA stimulated an increase in the tyrosine phosphorylation of focal adhesion kinase pp125 (pp125FAK) in EAhy 926 cells which was both protein kinase C- and pertussis toxin-independent. These results are discussed in terms of the pathways regulating both MAP kinase and pp125FAK in response to LPA in the EAhy 926 endothelial cells line.

Purinergic and cholinergic contractions in adult and neonatal rabbit bladder.

The contractile responses of isolated strips, of urinary bladder smooth muscle to various agonists and to nerve stimulation were compared in tissues taken from adult (greater than 14 weeks old) and neonatal (less than 1 week old) rabbits. There was no significant difference in the sensitivity of adult and neonatal tissues to a range of concentrations of acetylcholine (ACh) or carbachol, but in the neonatal tissues the contractile responses to ATP and to alpha, beta-methylene-ATP were significantly greater than in tissues from the adult. The contractile response to nerve stimulation was significantly greater in the neonatal tissues than in the adult. Experiments examining neurogenic contractions after atropine or desensitization of P2x purinoceptors suggest that the contribution of ATP and ACh to the contractile response changes with ageing.