Upregulation of p21(WAF1/Cip1) precedes tumor necrosis factor-induced necrosis-like cell death

The molecular mechanisms mediating death receptor-induced caspase-independent necrotic cell death are still largely unknown. We have previously reported that NIH3T3 cells are sensitized by caspase inhibition to death receptor-induced cytotoxicity leading to a necrosis-like cell death. In addition, we have identified an important role of cell cycle progression for this sensitization effect. Here, we report that tumor necrosis factor-induced necrotic death is preceded by an upregulation of the cyclin-dependent kinase inhibitor p21(WAF1/Cip1). Increased expression of p21(WAF1/Cip1) occurs prior to cell death in the nucleus, where it binds to a cyclin-dependent kinase indicating its functionality. The use of specific pharmacological inhibitors revealed a partial involvement of p38 mitogen-activated protein kinase in the upregulation of p21(WAF1/Cip1). Inhibition of p21(WAF1/Cip1) upregulation prevents a previously observed delay of the cells in the G2/M phase of the cell cycle thereby augmenting, not inhibiting cell death.     

Furosin, an ellagitannin, suppresses RANKL-induced osteoclast differentiation and function through inhibition of MAP kinase activation and actin ring formation

Phenolic compounds including tannins and flavonoids have been implicated in suppression of osteoclast differentiation/function and prevention of bone diseases. However, the effects of hydrolysable tannins on bone metabolism remain to be elucidated. In this study, we found that furosin, a hydrolysable tannin, markedly decreased the differentiation of both murine bone marrow mononuclear cells and Raw264.7 cells into osteoclasts, as revealed by the reduced number of tartrate resistant acid phosphatase (TRAP)-positive multinucleated cells and decreased TRAP activity. Furosin appears to target at the early stage of osteoclastic differentiation while having no cytotoxic effect on osteoclast precursors. Analysis of the inhibitory mechanisms of furosin revealed that it inhibited the receptor activator of nuclear factor-kappaB ligand (RANKL)-induced activation of p38 mitogen-activated protein kinase (p38MAPK) and c-Jun N-terminal kinase (JNK)/activating protein-1 (AP-1). Furthermore, furosin reduced resorption pit formation in osteoclasts, which was accompanied by disruption of the actin rings. Taken together, these results demonstrate that naturally occurring furosin has an inhibitory activity on both osteoclast differentiation and function through mechanisms involving inhibition of the RANKL-induced p38MAPK and JNK/AP-1 activation as well as actin ring formation.     

Apoptosis inhibitory activity of cytoplasmic p21(Cip1/WAF1) in monocytic differentiation

p21(Cip1/WAF1) inhibits cell-cycle progression by binding to G1 cyclin/CDK complexes and proliferating cell nuclear antigen (PCNA) through its N- and C-terminal domains, respectively. The cell-cycle inhibitory activity of p21(Cip1/WAF1) is correlated with its nuclear localization. Here, we report a novel cytoplasmic localization of p21(Cip1/WAF1) in peripheral blood monocytes (PBMs) and in U937 cells undergoing monocytic differentiation by in vitro treatment with vitamin D3 or ectopic expression of p21(Cip1/WAF1), and analyze the biological consequences of this cytoplasmic expression. U937 cells which exhibit nuclear p21(Cip1/WAF1) demonstrated G1 cell-cycle arrest and subsequently differentiated into monocytes. The latter event was associated with a cytoplasmic expression of nuclear p21(Cip1/WAF1), concomitantly with a resistance to various apoptogenic stimuli. Biochemical analysis showed that cytoplasmic p21(Cip1/WAF1) forms a complex with the apoptosis signal-regulating kinase 1 (ASK1) and inhibits stress-activated MAP kinase cascade. Expression of a deletion mutant of p21(Cip1/WAF1) lacking the nuclear localization signal (DeltaNLS-p21) did not induce cell cycle arrest nor monocytic differentiation, but led to an apoptosis-resistant phenotype, mediated by binding to and inhibition of the stress-activated ASK1 activity. Thus, cytoplasmic p21(Cip1/WAF1) itself acted as an inhibitor of apoptosis. Our findings highlight the different functional roles of p21(Cip1/WAF1), which are determined by its intracellular distribution and are dependent on the stage of differentiation.     

Tumor necrosis factor-alpha induces differentiation of and bone resorption by osteoclasts

Osteoclast progenitors differentiate into mature osteoclasts in the presence of receptor activator of NF-kappaB (RANK) ligand on stromal or osteoblastic cells and monocyte macrophage colony-stimulating factor (M-CSF). The soluble RANK ligand induces the same differentiation in vitro without stromal cells. Tumor necrosis factor-alpha (TNF-alpha), a potent cytokine involved in the regulation of osteoclast activity, promotes bone resorption via a primary effect on osteoblasts; however, it remains unclear whether TNF-alpha can also directly induce the differentiation of osteoclast progenitors into mature osteoclasts. This study revealed that TNF-alpha directly induced the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs), which produced resorption pits on bone in vitro in the presence of M-CSF. The bone resorption activity of TNF-alpha-induced MNCs was lower than that of soluble RANK ligand-induced MNCs; however, interleukin-1beta stimulated this activity of TNF-alpha-induced MNCs without an increase in the number of MNCs. In this case, interleukin-1beta did not induce TRAP-positive MNC formation. The osteoclast progenitors expressed TNF receptors, p55 and p75; and the induction of TRAP-positive MNCs by TNF-alpha was inhibited completely by an anti-p55 antibody and partially by an anti-p75 antibody. Our findings presented here are the first to indicate that TNF-alpha is a crucial differentiation factor for osteoclasts. Our results suggest that TNF-alpha and M-CSF play an important role in local osteolysis in chronic inflammatory diseases.     

Osteoclast differentiation is associated with transient upregulation of cyclin-dependent kinase inhibitors p21(WAF1/CIP1) and p27(KIP1)

Osteoclasts, bone-resorbing multinucleated cells, develop from monocyte-macrophage lineage cells in the presence of osteoclast differentiation factor (ODF, also called RANKL/TRANCE/OPGL) and macrophage colony-stimulating factor (M-CSF). M-CSF-dependent bone marrow macrophages (M-BMMPhis) from mouse bone marrow cells have been shown to differentiate into osteoclast-like multinucleated cells (OCLs) in the presence of soluble ODF/RANKL (sODF/RANKL) and M-CSF within 3 days. In this study, we found that stimulation of M-BMMPhis with sODF/RANKL induced a transient expression of cyclin-dependent kinase inhibitors (CDK inhibitors) p21(WAF1/CIP1) and p27(KIP1) by 24 h. The CDK inhibitor proteins disappeared by 48 h. Tumor necrosis factor alpha (TNF-alpha), which is reported to stimulate OCL differentiation, stimulated p21(WAF1/CIP1) and p27(KIP1) expression in M-BMMPhis as well. However, M-CSF alone did not stimulate the expression of the two CDK inhibitors. To clarify the role of p21(WAF1/CIP1) and p27(KIP1) in osteoclastogenesis, accumulation of these CDK inhibitors was aborted by antisense oligonucleotides. Treatment with p21(WAF1/CIP1) antisense oligonucleotide alone, or p27(KIP1) antisense oligonucleotide alone, showed a limited inhibitory effect on OCL formation. However, treatment with a mixture of these two antisense oligonucleotides strongly inhibited OCL formation. These results suggest that a combined modulation of the CDK inhibitors p21(WAF1/CIP1) and p27(KIP1) may be involved in osteoclast differentiation induced by ODF/RANKL.     

The Cyclin-Dependent Kinase Inhibitor p21CIP1/WAF1 Blocks Paclitaxel-Induced G2M Arrest and Attenuates Mitochondrial Injury and Apoptosis in p53-Null Human Leukemia Cells

The functional significance of the cyclin-dependent kinase inhibitor (CDKI) p21Cip1/WAF1 in paclitaxel-mediated lethality was examined in p53-null human leukemia cells (U937 and Jurkat). In these cells, paclitaxel exposure failed to induce p21Cip1/Waf1 expression. Nevertheless, stable expression of U937 cells with a p21Cip1/WAF1 antisense construct blocked paclitaxel-induced G2M arrest and significantly, albeit modestly, increased mitochondrial injury, caspase activation, apoptosis, and loss of clonogenic potential. These protective effects were less than those observed in cells exposed to the antimetabolite ara-C. Consistent with these results, enforced expression of p21Cip1/WAF1 in Jurkat cells transfected with a construct driven by a doxycycline-responsive promoter increased the percentage of cells arrested in G2M, but attenuated paclitaxel-mediated mitochondrial injury and apoptosis. Unexpectedly, enforced expression of p21Cip1/WAF1 diminished paclitaxel-mediated inactivation of ERK, and reduced paclitaxel-induced activation of JNK as well as Bcl-2 phosphorylation. Together, these findings suggest that the CDKI p21Cip1/WAF1 modestly but significantly protects p53-null human leukemia cells from paclitaxel-mediated lethality, and raise the possibility that p21Cip1/WAF1-associated perturbations in signal transduction pathways as well as Bcl-2 phosphorylation status may play a role in this phenomenon.     

Protein kinase B/Akt may regulate G2/M transition in the fertilized mouse egg by changing the localization of p21(Cip1/WAF1)

Protein kinase B (PKB, also called Akt) is known as a serine/threonine protein kinase. Some studies indicate that the Akt signalling pathway strongly promotes G2/M transition in mammalian cell cycle progression, but the mechanism remains to be clarified, especially in the fertilized mouse egg. Here, we report that the expression of Akt at both the protein and mRNA level was highest in G2 phase, accompanied by a peak of Akt activity. In addition, the subcellular localization of p21(Cip1/WAF1) has been proposed to be critical in the cell cycle. Hence, we detected the expression and localization of p21(Cip1/WAF1) after injecting fertilized mouse eggs with Akt mRNA. In one-cell stage fertilized embryos microinjected with mRNA coding for a constitutively active myristoylated Akt (myr-Akt), p21(Cip1/WAF1) was retained in the cytoplasm. Microinjection of mRNA of kinase-deficient Akt(Akt-KD) resulted in nuclear localization of p21(Cip1/WAF1) . Meanwhile, microinjection of different types of Akt mRNA affected the phosphorylation status of p21(Cip1/WAF1) . However, there was no obvious difference in the protein expression of p21(Cip1/WAF1) . Therefore, Akt controls the cell cycle by changing the subcellular localization of p21(Cip1/WAF1) , most likely by affecting the phosphorylation status of p21(Cip1/WAF1) .     

Cytoplasmic p21(Cip1/WAF1) regulates neurite remodeling by inhibiting Rho-kinase activity

p21(Cip1/WAF1) has cell cycle inhibitory activity by binding to and inhibiting both cyclin/Cdk kinases and proliferating cell nuclear antigen. Here we show that p21(Cip1/WAF1) is induced in the cytoplasm during the course of differentiation of chick retinal precursor cells and N1E-115 cells. Ectopic expression of p21(Cip1/WAF1) lacking the nuclear localization signal in N1E-115 cells and NIH3T3 cells affects the formation of actin structures, characteristic of inactivation of Rho. p21(Cip1/WAF1) forms a complex with Rho-kinase and inhibits its activity in vitro and in vivo. Neurite outgrowth and branching from the hippocampal neurons are promoted if p21(Cip1/WAF1) is expressed abundantly in the cytoplasm. These results suggest that cytoplasmic p21(Cip1/WAF1) may contribute to the developmental process of the newborn neurons that extend axons and dendrites into target regions.     

Interaction of Fas ligand and Fas expressed on osteoclast precursors increases osteoclastogenesis

We incidentally found that osteoclast precursors and mature osteoclasts express Fas ligand (FasL) as well as Fas, which was confirmed by flow cytometry, immunofluorescent staining, and RT-PCR. The aim of this study was to determine the role of FasL in differentiation and cell death of osteoclasts. To study the role of FasL in osteoclastogenesis, neutralizing anti-FasL mAb or rFasL was added during receptor activator of NF-kappaB ligand (RANKL)-induced osteoclastogenesis using bone marrow-derived macrophages. Neutralization of endogenous FasL by anti-FasL mAb decreased osteoclastogenesis, whereas rFasL enhanced osteoclast differentiation in a dose-dependent manner. In addition, rFasL up-regulated the secretion of osteoclastogenic cytokines, such as IL-1beta and TNF-alpha, and the activation of NF-kappaB. Functional blocking of IL-1beta and TNF-alpha using IL-1 receptor antagonist and soluble TNFR confirmed that those cytokines mediated the effect of FasL on osteoclastogenesis. The osteoclast precursors were relatively resistant to rFasL-induced apoptosis especially before RANKL treatment, resulting in minimal cell loss by rFasL treatment during osteoclastogenesis. Although rFasL increased the cell death of mature osteoclasts, growth factor withdrawal induced much more cell death. However, anti-FasL mAb did not affect the survival of mature osteoclasts, suggesting that the endogenous FasL does not have a role in the apoptosis of osteoclasts. Finally, in contrast to the effect on apoptosis, rFasL-assisted osteoclastogenesis was not mediated by caspases. In conclusion, FasL has a novel function in bone homeostasis by enhancing the differentiation of osteoclasts, which was not considered previously.     

G protein-coupled receptor 119 is involved in RANKL-induced osteoclast differentiation and fusion

G protein-coupled receptor 119 (GPR119) is known to be a promising therapeutic target for type 2 diabetes. Recently, it has been reported that the GPR119 agonist increases bone mineral density in an animal model of diabetes, suggesting that GPR119 may play a key role in bone metabolism. In this study, we investigated the functional role of GPR119 in receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation. We found that the GPR119 expression was markedly increased in preosteoclasts and then downregulated in mature osteoclasts. Activation of GPR119 with AS1269574, a potent selective agonist for GPR119, inhibited the generation of multinuclear osteoclasts from bone marrow-derived macrophages. Confirming this observation, targeted silencing of GPR119 using short hairpin RNA abrogated the AS1269574-mediated suppressive effect on osteoclast formation. GPR119 activation attenuated the expression of c-Fos and nuclear factor of activated T cells cytoplasmic 1 (NFATc1) and blocked RANKL-stimulated phosphorylation of IκBα, c-Jun N-terminal protein kinase (JNK), and extracellular signal-regulated kinase (ERK) but not p38. In addition, GPR119 activation suppressed preosteoclast fusion by downregulating the expression of the dendritic cell-specific transmembrane (DC-STAMP), a molecule that is essential for cell–cell fusion in osteoclast formation. Furthermore, ectopic expression of DC-STAMP restored AS1269574-mediated inhibition of osteoclast fusion. Taken together, our findings demonstrate that GPR119 plays a negative role in osteoclast differentiation and fusion induced by RANKL, and therefore may represent a potential target for bone resorption-associated diseases.     

Protein phosphatase 2Cγ regulates the level of p21 by Akt signaling

PP2Cγ is a splicing factor that dephosphorylates specific substrates required for the formation of the spliceosome. In a previous study, we reported that the degradation of p21^Cip1/WAF1was affected by PP2Cγ, causing an accumulation of cells in S phase. Here, we demonstrate that the PP2Cγ-induced degradation of p21^Cip1/WAF1 is mediated by Akt signaling. In cells expressing PP2Cγ, Akt1 protein was phosphorylated. When PP2Cγ expression was knocked down, the phosphorylation of Akt1 was reduced and the level of p21^Cip1/WAF1 protein was increased. Interestingly, the stability of p21^Cip1/WAF1 was highly maintained in Akt1-depleted cells despite the ectopic expression of PP2Cγ. Taken together, these results suggest that PP2Cγ is a novel regulator of p21^Cip1/WAF1 protein stability via the Akt signaling pathway.     

SEK1-dependent JNK1 activation prolongs cell survival during G-Rh2-induced apoptosis

We provide here evidence that c-Jun N-terminal protein kinase 1 (JNK1) activity is differentially up-regulated during apoptosis of SK-HEP-1 cells after treatment with ginsenoside Rh2 (G-Rh2). The G-Rh2-mediated JNK1 activation that occurred for the first 10-30min was associated with SEK1 activity, but thereafter, the sustained activation was associated not with SEK1 activity, but with proteolytic cleavage of JNK1-associated p21(WAF1/CIP1). Supporting this is that the expression of the dominant negative SEK1 mutant effectively blocked the early JNK1 activation phase but did not alter the sustained activation phase or apoptosis. Furthermore, expression of p21D112N, an uncleavable mutant of p21(WAF1/CIP1), suppressed the later JNK1 activation. Moreover, the stable overexpression of ectopic JNK1 suppressed apoptosis while expression of the dominant negative JNK1 mutant promoted it. We propose that the early SEK1-associated JNK1 activation phase acts to prolong cell survival in response to apoptosis-inducing agents, thereby serving as an intervening checkpoint prior to the commitment to apoptosis.     

A hybrid peptide derived from cecropin-A and magainin-2 inhibits osteoclast differentiation

The adult skeleton is in a dynamic state, being continually broken down and reformed by the coordinated actions of osteoclasts and osteoblasts. Increased osteoclast activity may contribute to the development of osteoporosis. Therefore, the intervention of osteoclast-mediated bone resorption is considered as an effective therapeutic approach in the treatment of osteoporosis. In the course of searching for agents that inhibit osteoclast differentiation and activation, we found that a novel hybrid peptide P1 derived from cecropin-A and magainin-2 reduced osteoclast differentiation in various osteoclast culture systems. As this peptide had no cytotoxicity on various cultures of primary cells and established cell lines, its inhibitory effect on osteoclastogenesis was not due to general cytotoxicity. The effects of P1 on osteoclasts appear to be mediated through the inhibition of NF-kappaB and JNK activation induced by the osteoclastogenic cytokine, receptor activator of NF-kappaB ligand (RANKL). These results provide an evidence for the potential usefulness of P1 for the treatment of bone-resorbing diseases.