Transcription-independent Mechanism Research Articles

Caspase cleavage of the APC tumor suppressor and release of an amino-terminal domain is required for the transcription-independent function of APC in apoptosis

The adenomatous polyposis coli (APC) tumor suppressor is inactivated by mutation in most colorectal tumors. APC is a component of the Wnt signaling pathway and is best known for its ability to downregulate beta-catenin and consequent effects on transcriptional regulation. Previous work demonstrated that APC accelerates apoptosis-associated caspase activity independently of transcription, and suggested novel tumor suppressor functions of APC. In this work, we have mapped the APC apoptosis-accelerating region to amino acids (aa) 1-760 by testing a series of non-overlapping APC segments. Interestingly, this segment corresponds to a stable group II caspase cleavage product of APC released during apoptosis that includes the amino-terminal aa1-777. Mutation of the APC aspartic acid residue at position 777 to an alanine completely abolished in vitro cleavage of APC by a recombinant group II caspase and rendered the full-length protein unable to accelerate apoptosis in vitro. A truncated APC protein associated with familial and sporadic colorectal cancer, also unable to accelerate apoptosis in vitro and in vivo, is resistant to group II caspase cleavage. These results demonstrate that cleavage of APC and the subsequent release of an amino-terminal segment are necessary for the transcription-independent mechanism of APC-mediated apoptosis.

Mitochondrial translocation of p53 underlies the selective death of hippocampal CA1 neurons after global cerebral ischaemia

p53, a tumour suppressor, is involved in DNA repair and cell death processes and mediates apoptosis in response to death stimuli by transcriptional activation of pro-apoptotic genes and by transcription-independent mechanisms. In the latter process, p53 induces permeabilization of the outer mitochondrial membrane by forming an inhibitory complex with a protective Bcl-2 family protein, resulting in cytochrome c release in several cell line systems. However, it is unclear how the mitochondrial p53 pathway mediates neuronal apoptosis after cerebral ischaemia. We examined interaction between the mitochondrial p53 pathway and vulnerable hippocampal CA1 neurons using a tGCI (transient global cerebral ischaemia) rat model. We showed mitochondrial translocation of p53 and its binding to Bcl-X(L). Mitochondrial p53 translocation, interaction between p53 and Bcl-X(L), and cytochrome c release from mitochondria and subsequent CA1 neuronal death were prevented by pifithrin-alpha, a p53-specific inhibitor. These results suggest that the mitochondrial p53 pathway plays a role in delayed CA1 neuronal death after tGCI.

Rapid Broad-Spectrum Analgesia through Activation of Peroxisome Proliferator-Activated Receptor-α

Severe pain remains a major area of unmet medical need. Here we report that agonists of the nuclear receptor PPAR-alpha (peroxisome proliferator-activated receptor-alpha) suppress pain behaviors induced in mice by chemical tissue injury, nerve damage, or inflammation. The PPAR-alpha agonists GW7647 [2-(4-(2-(1-cyclohexanebutyl)-3-cyclohexylureido)ethyl)phenylthio)-2-methylpropionic acid], Wy-14643 [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthioacetic acid], and palmitoylethanolamide (PEA) reduced nocifensive behaviors elicited in mice by intraplantar (i.pl.) injection of formalin or i.p. injection of magnesium sulfate. These effects were absent in PPAR-alpha-null mice yet occurred within minutes of agonist administration in wild-type mice, suggesting that they were mediated through a transcription-independent mechanism. Consistent with this hypothesis, blockade of calcium-operated IK(ca) (K(Ca)3.1) and BK(ca) (K(Ca)1.1) potassium channels prevented the effects of GW7647 and PEA in the formalin test. Three observations suggest that PPAR-alpha agonists may inhibit nocifensive responses by acting on peripheral PPAR-alpha. (i) PEA reduced formalin-induced pain at i.pl. doses that produced no increase in systemic PEA levels; (ii) PPAR-alpha was expressed in dorsal root ganglia neurons of wild-type but not PPAR-alpha-null mice; and (ii) GW7647 and PEA prevented formalin-induced firing of spinal cord nociceptive neurons in rats. In addition to modulating nociception, GW7647 and PEA reduced hyperalgesic responses in the chronic constriction injury model of neuropathic pain; these effects were also contingent on PPAR-alpha expression and were observed following either acute or subchronic PPAR-alpha agonist administration. Finally, acute administration of GW7647 and PEA reduced hyperalgesic responses in the complete Freund's adjuvant and carrageenan models of inflammatory pain. Our results suggest that PPAR-alpha agonists may represent a novel class of analgesics.

Mdm2 inhibitor Nutlin-3a induces p53-mediated apoptosis by transcription-dependent and transcription-independent mechanisms and may overcome Atm-mediated resistance to fludarabine in chronic lymphocytic leukemia

AbstractAlthough TP53 mutations are rare in B-cell chronic lymphocytic leukemia (CLL), Mdm2 overexpression has been reported as an alternative cause of p53 dysfunction. We investigated the potential therapeutic use of nongenotoxic p53 activation by a small-molecule antagonist of Mdm2, Nutlin-3a, in CLL. Nutlin-3a induced significant apoptosis in 30 (91%) of 33 samples from previously untreated patients with CLL; all resistant samples had TP53 mutations. Low levels of Atm (ataxia telangiectasia mutated) or high levels of Mdm2 (murine double minute 2) did not prevent Nutlin-3a from inducing apoptosis. Nutlin-3a used transcription-dependent and transcription-independent pathways to induce p53-mediated apoptosis. Predominant activation of the transcription-independent pathway induced more pronounced apoptosis than that of the transcription-dependent pathway, suggesting that activation of the transcription-independent pathway is sufficient to initiate p53-mediated apoptosis in CLL. Combination treatment of Nutlin-3a and fludarabine synergistically increased p53 levels, and induced conformational change of Bax and apoptosis in wild-type p53 cells but not in cells with mutant p53. The synergistic apoptotic effect was maintained in samples with low Atm that were fludarabine resistant. Results suggest that the nongenotoxic activation of p53 by targeting the Mdm2-p53 interaction provides a novel therapeutic strategy for CLL.

Raloxifene Increases Proliferation and Up-regulates Telomerase Activity in Human Umbilical Vein Endothelial Cells

Vascular endothelial senescence is involved in human atherosclerosis. Telomerase activity is known to be critical in cellular senescence and its level is modulated by regulation of telomerase catalytic subunit (telomerase reverse transcriptase (TERT)) at both the transcriptional and post-transcriptional levels. Since the cardioprotective effect of estrogen itself has not been ruled out, we examined that of raloxifene, which has been classified as a selective estrogen receptor modulator, on the proliferation and telomerase activity of human umbilical vein endothelial cells (HUVECs). Raloxifene, like estrogen, clearly induced the telomerase activity and human TERT (hTERT) expression via estrogen receptor (ER) alpha and ERbeta. Treatment with raloxifene for 5 days significantly induced cell growth, and either cotreatment with a telomerase inhibitor, 3'-azido-3'-deoxythymidine, or transfection with hTERT-specific small interfering RNA significantly attenuated the raloxifene-induced cell growth. Raloxifene also induced the phosphorylation of Akt, and pretreatment with a phosphatidylinositol 3-kinase inhibitor, LY294002, significantly attenuated the raloxifene-induced telomerase activity. In addition, raloxifene induced both the phosphorylation of hTERT and IkappaB. Moreover, cotreatment with an IkappaBalpha phosphorylation inhibitor, BAY-11-7082, or a specific NFkappaB nuclear translocation inhibitor, SN50, significantly attenuated the raloxifene-induced telomerase activity and the association of NFkappaB with hTERT. These results show that raloxifene induced the up-regulation of telomerase activity not only by the transcriptional regulation of hTERT but also by post-translational regulation of the phosphorylation of Akt and hTERT and the association of hTERT with NFkappaB in HUVECs. Thus, the up-regulation of telomerase activity in vascular endothelial cells might be one mechanism contributing to the potential atheroprotective effect of raloxifene.

Kinome Analysis Reveals Nongenomic Glucocorticoid Receptor-Dependent Inhibition of Insulin Signaling

Glucocorticoids (GCs) are powerful immunosuppressive agents that control genomic effects through GC receptor (GR)-dependent transcriptional changes. A common complication of GC therapy is insulin resistance, but the underlying molecular mechanism remains obscure. Evidence is increasing for rapid genomic-independent GC action on cellular physiology. Here, we generate a comprehensive description of nongenomic GC effects on insulin signaling using peptide arrays containing 1,176 different kinase consensus substrates. Reduced kinase activities of the insulin receptor (INSR) and several downstream INSR signaling intermediates (i.e. p70S6k, AMP-activated protein kinase, glycogen synthase kinase-3, and Fyn) were detected in adipocytes and T lymphocytes due to short-term treatment with dexamethasone (DEX), a synthetic fluorinated GC. Western blot analysis confirmed suppressed phosphorylation of the INSR and a series of downstream INSR targets (i.e. INSR substrate-1, p70S6k, protein kinase B, phosphoinositide-dependent protein kinase, Fyn, and glycogen synthase kinase-3) after DEX treatment. DEX inhibited insulin signaling through a GR-dependent (RU486 sensitive) and transcription-independent (actinomycin D insensitive) mechanism. Overall, we postulate here a molecular mechanism for GC-induced insulin resistance based on nongenomic GR-dependent inhibition of insulin signaling.

Restoring p53-mediated apoptosis in cancer cells: New opportunities for cancer therapy

The p53 gene is the most commonly mutated gene known in human tumors; over half of human tumors contain inactivating mutations in p53. In the past decade, the role of p53 as apoptotic trigger has been well demonstrated both in vitro and in vivo. Many chemotherapeutic agents cause DNA damage and activate the p53 pathway to induce growth arrest and apoptosis. However, the p53 function is often inactivated or suppressed in human cancers. Thus, functional restoration of this pathway is an attractive therapeutic strategy. In recent years, a number of therapeutic approaches aiming at modulation of the p53 pathway have been developed and will be reviewed here. The focus will be on recent developments elucidating a transcription-independent mechanism of p53-mediated apoptosis and the therapeutic opportunities arising from this new mechanism.

Targeting histone deacetylase in cancer therapy

Histone deacetylase (HDAC) is recognized as one of the promising targets for cancer treatment as many HDAC inhibitors have entered clinical trials for both solid and liquid tumors. Nevertheless, the mechanisms underlying the antiproliferative effects of HDAC inhibitors remain elusive. Although they have been shown to regulate the transcription of a defined set of genes through chromatin remodeling, increasing evidence suggests that modifications of the epigenetic histone code may not be the primary mechanism for HDAC inhibitor-mediated growth inhibition and apoptosis in cancer cells. While histones still represent a primary target for the physiological function of HDACs, the antitumor effect of HDAC inhibitors might also be attributed to transcription-independent mechanisms by modulating the acetylation status of a series of nonhistone targets. Also noteworthy is the effect of HDAC inhibitors on Akt downregulation through the alteration of protein phosphatase 1 (PP1) complex formation. To provide an overview of the use of HDAC inhibitors in cancer treatment, this review addresses the following subjects: (1) the physiological relevance of HDAC-mediated acetylation of histone and nonhistone substrates, (2) the chemical biology of HDACs and development of a novel class of HDAC inhibitors, and (3) the protein acetylation-independent effect of HDAC inhibitors on the activation status of signaling kinases.

The N-terminal domain of tumor suppressor p53 is involved in the molecular interaction with the anti-apoptotic protein Bcl-Xl

Emerging evidences suggest that transcription-independent mechanism of p53 appears to make an important contribution to the overall p53-dependent apoptosis. Recently, it has been postulated that the DNA-binding domain of p53 can interact with Bcl-Xl, and subsequently the proposed molecular interaction has been shown by NMR studies. Interestingly, Chipuk et al. [Cancer Cell 4 (2003) 371] reported that the N-terminal domain of p53 (p53NTD) alone is necessary and sufficient to induce transcription-independent apoptosis. To further define and understand the nature of the molecular recognition between p53 and Bcl-Xl, our current study focuses on p53NTD. We first demonstrated the molecular interaction between p53NTD and Bcl-Xl by co-expressing and purifying the complex. Second, to define the binding interface of the molecular interaction, which is not previously characterized, in the current we employed a NMR-based binding study, showing that the binding site on Bcl-Xl is located in the region including α4, the N- and C-termini of α3, the N-terminus of α5, and the central part of α2. To further probe this observation, we then performed fluorescence resonance energy transfer (FRET) assay in cells. The FRET efficiency detected between the donor and acceptor molecules appears to suggest the presence of molecular interaction of p53NTD with Bcl-Xl in cells. Taken together, our data suggest that p53NTD interacts with Bcl-Xl but the characteristic of the molecular interaction appears to be different from that of the DNA-binding domain of p53.

Effects of exogenous p53 transfection on the gene expression in the human brain glioma cell line U251

The p53 gene is activated in response to several malignancy-associated stress signals by transactivation of downstream genes and by transcription-independent mechanisms. In order to identify new p53 downstream genes, we established a new system of p53 gene inducible expression, U251-pTet-p53 cell line, with the Tet-On Gene Expression System, in which exogenous p53 gene could overexpress in doxycycline (Dox) medium but not in the medium without Dox. By comparing their random primer RT-PCR products, it was proved that exogenous p53 gene expression could lead to many genes differential expression, some up-expressed and others down-expressed. All of these differential expressed genes may be p53 downstream genes. We can gain the magnitude of p53 downstream genes, which provides the basis of directly cloning of novel p53 downstream genes and further studying of p53 regulatory network.

Antagonistic Functions of Tetradecanoyl Phorbol Acetate-Inducible-Sequence 11b and HuR in the Hormonal Regulation of Vascular Endothelial Growth Factor Messenger Ribonucleic Acid Stability by Adrenocorticotropin

Expression of vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen and a potent angiogenic factor, is up-regulated by a variety of factors including hypoxia, growth factors, and hormones. In the adrenal cortex, regulation of VEGF expression by the pituitary hormone ACTH ensures the maintenance of the organ vasculature. We have previously shown that ACTH evokes a rapid and transient increase in VEGF mRNA levels in primary adrenocortical cells through transcription-independent mechanisms. We further demonstrated that the zinc finger RNA-binding protein Tis11b (tetradecanoyl phorbol acetate-inducible-sequence 11b) destabilizes VEGF mRNA through its 3'-untranslated region (3'-UTR) and that Tis11b is involved in the decay phase of ACTH-induced VEGF mRNA expression. In the present study, we attempted to determine the mechanisms underlying ACTH-elicited increase in VEGF mRNA levels in adrenocortical cells. We show that ACTH triggers an increase in the levels of the mRNA-stabilizing protein HuR in the cytoplasm and a concomitant decrease in the levels of HuR in the nucleus. This process is accompanied by an increased association of HuR with the nucleocytoplasmic shuttling protein pp32, indicating that ACTH induces HuR translocation from the nuclear to the cytoplasmic compartment. Leptomycin B, a specific inhibitor of CRM1-dependent nuclear export of pp32, significantly reduced ACTH-induced VEGF mRNA levels. Furthermore, RNA interference-mediated depletion of HuR in adrenocortical cells abrogated ACTH-induced VEGF mRNA expression. Finally, we show that Tis11b and HuR exert antagonistic effects on VEGF 3'-UTR in vitro. Although both proteins could bind simultaneously on VEGF 3'-UTR, Tis11b markedly decreases HuR-binding to this RNA sequence. Altogether, these results suggest that the RNA-stabilizing protein HuR is instrumental to ACTH-induced expression of VEGF mRNA and that the nuclear export of HuR is a rate-limiting step in this process. HuR appears to transiently stabilize VEGF transcripts after ACTH stimulation of adrenocortical cells, and Tis11b appears to subsequently trigger their degradation.

Mitochondrially Targeted p53 Has Tumor Suppressor Activities In vivo

Complex proapoptotic functions are essential for the tumor suppressor activity of p53. We recently described a novel transcription-independent mechanism that involves a rapid proapoptotic action of p53 at the mitochondria and executes the shortest known circuitry of p53 death signaling. Here, we examine if this p53-dependent mitochondrial program could be exploited for tumor suppression in vivo. To test this, we engage Emu-Myc transgenic mice, a well-established model of p53-dependent lymphomagenesis. We show that exclusive delivery of p53 to the outer mitochondrial membrane confers a significant growth disadvantage on Emu-Myc-transformed B-cells of p53-deficient or alternate reading frame-deficient genotypes, resulting in efficient induction of apoptosis and impinged proliferation. Conversely, normal cells from thymus, spleen, and bone marrow showed poor infectivity with these viruses. This proof-of-principle experiment shows that exclusive reliance on the direct mitochondrial program exerts a significant tumor suppressor activity in vivo. Our in vivo data on the direct mitochondrial apoptotic p53 program lays the groundwork to further investigate its efficacy and safety and to address its possible therapeutic value in the future.

Irradiation-induced Translocation of p53 to Mitochondria in the Absence of Apoptosis

The tumor suppressor protein p53 promotes apoptosis in response to death stimuli by transactivation of target genes and by transcription-independent mechanisms. Recently, it was shown that during apoptosis p53 can specifically translocate to mitochondria, where it physically interacts with and inactivates prosurvival Bcl-2 proteins. In the present study, we therefore investigated the role of mitochondrial translocation of p53 for the stress response of tumor cells. In various cell lines, DNA damage induced by either ionizing irradiation or topoisomerase inhibitors triggered a robust translocation of a fraction of p53 to mitochondria to a similar extent. Nevertheless, the cells succumbed to apoptosis only in response to topoisomerase inhibitors, but remained resistant to apoptosis induced by ionizing radiation. Irradiated cells became senescent, although irradiation triggered a functional p53 response and induced expression of p21, Bax, and Puma. Interestingly, even the targeted expression of p53 to mitochondria was insufficient to launch apoptosis, whereas overexpression of wild-type p53 induced Bax activation and apoptotic alterations. Together, these results suggest that, in contrast to previous reports, mitochondrial translocation of p53 does not per se lead to cell death and that this might constitute a mechanism that contributes to the resistance of tumor cells to ionizing radiation-induced apoptosis.

Pharmacologic Modulation of Glycogen Synthase Kinase-3β Promotes p53-Dependent Apoptosis through a Direct Bax-Mediated Mitochondrial Pathway in Colorectal Cancer Cells

Activation of p53 tumor suppressor induces either cell cycle arrest or apoptosis through transcription-dependent and independent pathways; however, their relative roles in apoptosis induction and how these pathways are regulated remains elusive. Here, we report a unique role for glycogen synthesis kinase-3beta (GSK-3beta) in regulating p53 functions in human colorectal cancer cells. Pharmacologic modulation of GSK-3beta markedly impaired p53-dependent transactivation of targets including p21 and Puma but promoted p53-dependent conformational activation of Bax, resulting in cytochrome c release, loss of mitochondrial membrane potential, and caspase-9 processing. Thus, p53-mediated damage response is converted from cell cycle arrest to apoptosis following exposure to a variety of chemotherapeutic agents. We found that this effect is associated with the modulation of inhibitory Ser(9) phosphorylation of GSK-3beta but not with the activating tyrosine phosphorylation. We further show that the induction of apoptosis is through a direct mitochondrial pathway that requires Bax but not Puma. Our results underscore the importance of transcription-independent mechanism in p53-induced apoptosis and indicate that GSK-3beta plays distinct dual roles in regulating p53 pathways: promoting p53 transcriptional activity in the nucleus but suppressing p53-mediated direct apoptotic function at the mitochondria. Importantly, our data suggest that small-molecule inhibition of GSK-3beta might represent a novel approach for modulating chemotherapy.

Inhibition of c-Jun-N-terminal-Kinase Sensitizes Tumor Cells to CD95-Induced Apoptosis and Induces G2/M Cell Cycle Arrest

Loss of susceptibility to apoptosis signals is a crucial step in carcinogenesis. Therefore, sensitization of tumor cells to apoptosis is a promising therapeutic strategy. c-Jun-N-terminal-kinases (JNK) have been implicated in stress-induced apoptosis, but may also contribute to survival signaling. Here we show that CD95-induced apoptosis is augmented by the JNK inhibitor SP600125 and small interfering RNA directed against JNK1/2. SP600125 potently inhibited methyl methane sulfonate-induced phosphorylation of c-Jun, but had minimal effect on apoptosis alone. In contrast, it strongly enhanced CD95-mediated apoptosis in six of eight tumor cell lines and led to a G2/M phase arrest in all cell lines. SP600125 enhanced cleavage of caspase 3 and caspase 8, the most upstream caspase in the CD95 pathway. JNK inhibition up-regulates p53 and its target genes p21Cip1/Waf1 and CD95. However, although HCT116 p53-/- cells and p21+/+ cells were less sensitive to CD95 stimulation than their p53+/+ and p21-/- counterparts, p53 and p21 were not involved in the JNK-mediated effect. JunD, which was described to be protective in tumor necrosis factor-induced apoptosis, was not regulated by JNK inhibition on the protein level. When transcription was blocked by actinomycin D, JNK inhibition still enhanced apoptosis to a comparable extent. We conclude that JNK inhibition has antitumor activity by inducing growth arrest and enhancing CD95-mediated apoptosis by a transcription-independent mechanism.

Mitochondrial Ribosomal Protein L41 Suppresses Cell Growth in Association with p53 and p27Kip1

The p53 protein arrests the cell cycle at the G1 phase when stabilized by the interaction between ribosomal proteins and HDM2 under growth-inhibitory conditions. Meanwhile, p53, when translocated to the mitochondria in response to cell death signals, induces apoptosis via transcription-independent mechanisms. In this report, we demonstrate that the mitochondrial ribosomal protein L41 (MRPL41) enhances p53 stability and contributes to p53-induced apoptosis in response to growth-inhibitory conditions such as actinomycin D treatment and serum starvation. An analysis of MRPL41 expression in paired normal and tumor tissues revealed lower expression in tumor tissue. Ectopic MRPL41 expression resulted in inhibition of the growth of cancer cells in tissue culture and tumor growth in nude mice. We discovered that MRPL41 protein is localized in the mitochondria, stabilizes the p53 protein, and enhances its translocation to the mitochondria, thereby inducing apoptosis. Interestingly, in the absence of p53, MRPL41 stabilizes the p27(Kip1) protein and arrests the cell cycle at the G1 phase. These results suggest that MRPL41 plays an important role in p53-induced mitochondrion-dependent apoptosis and MRPL41 exerts a tumor-suppressive effect in association with p53 and p27 (Kip1).

Chk2 regulates transcription-independent p53-mediated apoptosis in response to DNA damage

The tumor suppressor protein p53 plays a central role in the induction of apoptosis in response to genotoxic stress. The protein kinase Chk2 is an important regulator of p53 function in mammalian cells exposed to ionizing radiation (IR). Cells derived from Chk2-deficient mice are resistant to the induction of apoptosis by IR, and this resistance has been thought to be a result of the defective transcriptional activation of p53 target genes. It was recently shown, however, that p53 itself and histone H1.2 translocate to mitochondria and thereby induces apoptosis in a transcription-independent manner in response to IR. We have now examined whether Chk2 also regulates the transcription-independent induction of apoptosis by p53 and histone H1.2. The reduced ability of IR to induce p53 stabilization in Chk2-deficient thymocytes was associated with a marked impairment of p53 and histone H1 translocation to mitochondria. These results suggest that Chk2 regulates the transcription-independent mechanism of p53-mediated apoptosis by inducing stabilization of p53 in response to IR.

Rapid, Membrane-Initiated Actions of 1,25 Dihydroxyvitamin D: What Are They and What Do They Mean?

Vitamin D is a conditionally required nutrient traditionally thought to influence physiology as the metabolite 1,25-dihydroxyvitamin D [1,25(OH)(2) D] by binding to the vitamin D receptor (VDR) and stimulating the transcription of genes through direct VDR-DNA interactions. However, over the past 15 y research has demonstrated that 1,25(OH)(2) D, as well as other steroid hormones, can rapidly stimulate ion fluxes and activate protein kinases by transcription-independent mechanisms. This review summarizes recent research on the rapid actions of 1,25(OH)(2) D and identifies questions that remain to be answered in this area.

Mechanisms of Mitogen-Activated Protein Kinase Inhibition by Parathyroid Hormone in Osteoblast-Like Cells

Parathyroid hormone (PTH) dose dependently inhibits growth factor- and stress-induced osteoblast proliferation via inactivating mitogen-activated protein kinase (MAPK) signaling pathways. Osteoblasts have recently been shown to express MAPK phosphatase (MKP)-1, a dual-specific phosphatase inactivator of MAPK. Investigated was the role of MKPs in the PTH-induced attenuation of MAPK and Jun N-terminal kinase (JNK) signaling in osteoblast-like UMR106-01 cells. PTH induced a persistent inhibition of p42/44 MAPK and JNK phosphorylation starting at 10 min of incubation and lasting for at least 2 h. Actinomycin D affected both p42/44 MAPK and JNK dephosphorylation by PTH, suggesting a transcription-dependent mechanism of action. PTH rapidly and transiently induced expression of MKP-1. MKP-1 mRNA was already elevated after 10 min of 10(-7) M PTH incubation, reached maximal expression after 30 to 60 min, and remained elevated after 4 h. MKP-1 protein was also upregulated within 30 to 60 min of PTH administration. The protein kinase A inhibitor H89 partly reduced PTH-induced MKP-1 expression, but the protein kinase C inhibitor bisindolylmaleimide had no effect, suggesting that PTH induces MKP-1 mainly via the protein kinase A pathway. MKP-2 mRNA was downregulated after 2 h after an early period of induction, and MKP-3 mRNA was immediately reduced. Ro 318-220 did not affect PTH-induced MAPK inactivation but effectively blocked JNK dephosphorylation. The time course of PTH-induced MKP-1 protein expression closely correlated with JNK dephosphorylation. PTH attenuates the stress-induced JNK signaling pathway in osteoblasts via induction of MKP-1 synthesis but inhibits the p42/44 MAPK pathway mainly via transcription-independent mechanisms.

Transcriptional repression by p53 promotes a Bcl-2-insensitive and mitochondria-independent pathway of apoptosis.

p53 can induce apoptosis in various ways including transactivation, transrepression and transcription-independent mechanisms. What determines the choice between them is poorly understood. In a rat embryo fibroblast model, caspase inhibition changed the outcome of p53 activation from standard Bcl-2-regulated apoptosis to caspase-independent and Bcl-2-insensitive cell death, a phenomenon not described previously. Here, we show that caspase inhibition affects cell death commitment decisions by modulating the apoptotic functions of p53. Indeed, in the Bcl-2-sensitive pathway, transactivation-dependent signalling is activated leading to a rapid MDM2-mediated degradation of p53. In contrast, in the Bcl-2-insensitive pathway, p53 is stable and this is associated with transrepression-dependent signalling. A study with microarrays identified these genes regulated by p53 in the absence of active caspases.