p14ARF inhibits the growth of p53 deficient cells in a cell-specific manner.

While p14(ARF) suppression of tumorigenesis in a p53-dependent manner is well studied, the mechanism by which p14(ARF) inhibits tumorigenesis independently of p53 remains elusive. A variety of factors have been reported to play a role in this latter process. We report here that p14(ARF) displays different effects on the anchorage-dependent and -independent growth of p53-null/Mdm2 wild type cells. p14(ARF) blocks both the anchorage-dependent and-independent (soft agar) proliferation of 293T and p53(-/-) HCT116, but not p53-null H1299 lung carcinoma cells. While p14(ARF) had no effect on the anchorage-dependent proliferation of p53(-/-) MEFs and Ras12V-transformed p53(-/-) MEFs, it inhibited the growth of Ras12V-transformed p53(-/-) MEFs in soft agar. Furthermore, ectopic expression of p14(ARF) did not lead to degradation of the E2F1 protein and did not result in the reduction of E2F1 activity detected by two E2F1 responsible promoters, Apaf1 and p14(ARF) promoter, in 293T, p53(-/-) HCT116, and H1299 cells. This is consistent with our observations that p14(ARF) did not result in G1 arrest, but induced apoptosis via Bax up-regulation. Taken together, our data demonstrate that the response of p53-null cells to ARF is cell type dependent and involves factors other than Mdm2 and E2F1.

Akt mediates mechanical strain-induced collagen production by mesangial cells.

Increased glomerular hydrostatic pressure is an important determinant of glomerulosclerosis and can be modeled by in vitro exposure of mesangial cells to cyclic mechanical strain. Stretched mesangial cells increase extracellular matrix protein production, the hallmark of glomerulosclerosis. Recent data indicate that the serine/threonine kinase Akt may be involved in matrix modulation. Thus, Akt activation and matrix synthesis in stretched mesangial cells were studied. Exposure of mesangial cells to 1 Hz cyclic strain led to prompt Akt activation, which was biphasic to 24 h. Activation was dependent on signaling through phosphatidylinositol-3-kinase and required EGF receptor transactivation. Inhibition of signaling through the PDGF receptor, Src kinase, or cytoskeletal disruption failed to prevent strain-induced Akt activation. Collagen type 1A1 transcript expression, promoter activation, and protein secretion were increased by stretch at 24 h and were dependent on phosphatidylinositol-3 kinase. Overexpression of dominant-negative Akt inhibited strain-induced collagen 1A1 production. Conversely, overexpression of constitutively active Akt led to increased collagen 1A1 upregulation and secretion. Finally, Akt activation was observed in the glomeruli of remnant rat kidneys, a model marked by increased intraglomerular pressure. The authors conclude that mechanical strain induces Akt activation in mesangial cells through a mechanism requiring phosphatidylinositol-3-kinase and EGF receptor transactivation. Type 1 collagen production is dependent on Akt and can be induced by Akt overexpression. Akt activation is observed in remnant kidneys in vivo. Thus, the role of Akt in progression of chronic hemodynamic glomerular disease is worthy of further exploration.

Stretch-induced Raf-1 activation in mesangial cells requires actin cytoskeletal integrity.

Glomerular capillary hypertension is a determinant of glomerulosclerosis and is modelled in vitro by exposure of mesangial cells to cyclic mechanical strain. In response to strain, Erk is activated and mediates extracellular matrix accumulation and mesangial cell proliferation. Erk activation is dependent on an intact cytoskeleton. Since Raf-1 lies upstream of Erk in response to numerous stimuli, and since its activation is dependent on membrane recruitment, we postulated that the cytoskeleton was essential for Raf-1 membrane recruitment and Erk activation. Primary rat mesangial cells (passages 8-20) were stretched at 1 Hz and 27 kPa. Raf-1 was both phosphorylated on serine-338 (S338) and activated within 2 min of strain. The Raf-1 inhibitor, GW5074, dose-dependently blocked strain-induced Erk activation and Raf-1 phosphorylation. Although phosphatidylinositol-3-kinase (PI3-K) may mediate Raf-1 activation, PI3-K inhibition with wortmannin or LY294002 had no effect on stretch-induced Raf-1 activation. Cytoskeletal disruption with cytochalasin D and the Rho-kinase inhibitor, Y-27632, however, blocked both Raf-1 phosphorylation and activation. Furthermore, membrane localization of Raf-1 was increased by strain and prevented by cytoskeletal disruption. Thus, strain leads to rapid membrane localization, S338 phosphorylation, and activation of Raf-1. These events are independent of PI3-K, but require Rho-kinase activation and an intact actin cytoskeleton.

ATM activity contributes to the tumor-suppressing functions of p14ARF.

P14/p19ARF (ARF) plays a major role in the activation of p53 by oncogenic signals. The biochemical basis of this has not been fully elucidated. We report here that forced expression of p14ARF enhances phosphorylation of p53 serine 15 (p53S15) in NIH3T3, IMR90 and MCF7 cells. Ectopic expression of the oncogenes c-myc, E2F1 and E1A, all of which activate p53 at least partially via ARF, lead to p53S15 phosphorylation in IMR90 cells. In addition, ectopic expression of p53 also results in p53S15 phosphorylation, suggesting that this is a common event in the ARF-p53 tumor suppression system. Furthermore, p53-, p14ARF-, c-myc- and E2F1-, but not E1A-, induced p53S15 phosphorylation was substantially reduced in AT fibroblasts (GM05823). Downregulation of ATM in MCF7 cells using RNA interference (RNAi) technology significantly attenuated p14ARF- and p53-induced phosphorylation of p53S15. Ectopically expressed ARF in NIH3T3 cells induced ATM nuclear foci and activated ATM kinase. Functionally, ectopic expression of p14ARF and c-myc inhibited the proliferation of IMR90 but not ATM null GM05823 cells, and p14ARF-induced inhibition of MCF7 cell proliferation was significantly attenuated by downregulation of ATM by RNAi. Taken together, these data show a functional role for ATM in ARF-mediated tumor suppression.

Nitric oxide inhibits stretch-induced MAPK activation in mesangial cells through RhoA inactivation.

Glomerular capillary hypertension is an important determinant of glomerulosclerosis in rats with subtotal renal ablation. Dietary supplementation with L-arginine increases renal nitric oxide (NO) production and limits glomerular injury in this model, and early benefits are seen without altered glomerular capillary pressure. In an in vitro model of hemodynamically mediated signaling, the authors have reported that subjecting MC to cyclic stretch/relaxation activates the mitogen-activated protein kinase p42/44 (Erk) cascade and that NO and cyclic GMP abrogate stretch-induced Erk activation by inducing actin cytoskeletal disassembly. The actin cytoskeleton is regulated by the Rho family of GTPases, including RhoA; therefore, the authors examined the role of RhoA in stretch-induced Erk activation and as an NO target. In primary rat MC subjected to cyclic mechanical strain, RhoA activity was maximally increased (2.4-fold) after 1 min of stretch, and Erk activation temporally followed. The Rho-kinase inhibitor Y-27632 attenuated Erk activation in a dose-dependent manner and prevented stretch-induced actin stress fiber formation. The NO donors S-nitroso-N-acetylpenicillamine and cGMP both inhibited stretch-induced RhoA and Erk activation and stress fiber formation. Infection of MC with the RhoA mutant RhoA-Ala188, which is resistant to NO-dependent phosphorylation, abrogated the effects of NO and cGMP on stretch-induced Erk activation and stress fiber formation. The authors conclude that the early activation of RhoA is essential for stretch-induced actin stress fiber formation and Erk activation in MC, events which are prevented by NO and cGMP through their action on RhoA. Inhibition of RhoA may thus be a new approach to the prevention of hemodynamically mediated glomerular injury.

ERK activation mediates cell cycle arrest and apoptosis after DNA damage independently of p53.

In response to DNA damage, ataxia-telangiectasia mutant and ataxia-telangiectasia and Rad-3 activate p53, resulting in either cell cycle arrest or apoptosis. We report here that DNA damage stimuli, including etoposide (ETOP), adriamycin (ADR), ionizing irradiation (IR), and ultraviolet irradiation (UV) activate ERK1/2 (ERK) mitogen-activated protein kinase in primary (MEF and IMR90), immortalized (NIH3T3) and transformed (MCF-7) cells. ERK activation in response to ETOP was abolished in ATM-/- fibroblasts (GM05823) and was independent of p53. The MEK1 inhibitor PD98059 prevented ERK activation but not p53 stabilization. Maximal ERK activation in response to DNA damage was not attenuated in MEF(p53-/-). However, ERK activation contributes to either cell cycle arrest or apoptosis in response to low or high intensity DNA insults, respectively. Inhibition of ERK activation by PD98059 or U0126 attenuated p21(CIP1) induction, resulting in partial release of the G(2)/M cell cycle arrest induced by ETOP. Furthermore, PD98059 or U0126 also strongly attenuated apoptosis induced by high dose ETOP, ADR, or UV. Conversely, enforced activation of ERK by overexpression of MEK-1/Q56P sensitized cells to DNA damage-induced apoptosis. Taken together, these results indicate that DNA damage activates parallel ERK and p53 pathways in an ATM-dependent manner. These pathways might function cooperatively in cell cycle arrest and apoptosis.

Apoptotic release of histones from nucleosomes.

Chromatin structure is influenced by histone modification, and this may help direct chromatin behavior to facilitate transcription, DNA replication, and DNA repair. Chromatin condensation and DNA fragmentation are the classic nuclear features but remain poorly characterized. It is highly probable that nucleosomal structure must be altered to allow these features to become apparent, but data to support this construct are lacking. We report here that in response to apoptotic signals from a death receptor (CD95 and tumor necrosis factor-alpha) or mitochondrial (staurosporine) apoptotic stimulus, the core nucleosomal histones H2A, H2B, H3, and H4 become separated from DNA during apoptosis in Jurkat and HeLa cells and are consequently detectable in the cell lysate prepared using a non-ionic detergent. The timing of this histone release from DNA correlates well with the progression of apoptosis. We also show expression of a caspase cleavage-resistant form of ICAD (ICAD-DM) in Jurkat and HeLa cells abolished DNA fragmentation and also dramatically reduced histone release in apoptotic cells. However, we demonstrate that apoptotic histone release is not an inevitable consequence of CAD/DFF-40-mediated DNA destruction as DNA fragmentation but not histone release occurs efficiently in tumor necrosis factor-alpha- and etoposide-treated NIH3T3 cells. Furthermore, in an in vitro apoptotic assay, incubation of apoptotic Jurkat cellular extract with non-apoptotic Jurkat nuclei led to nuclear DNA fragmentation without obvious histone release. Taken together, these data demonstrate that CAD/DFF-40 functions indirectly in mediating nucleosomal destruction during apoptosis.