Tissue properties of the human vertebral body sub-structures evaluated by means of microindentation.

PURPOSE: The better understanding of vertebral mechanical properties can help to improve the diagnosis of vertebral fractures. As the bone mechanical competence depends not only from bone mineral density (BMD) but also from bone quality, the goal of the present study was to investigate the anisotropic indentation moduli of the different sub-structures of the healthy human vertebral body and spondylophytes by means of microindentation. METHODS: Six human vertebral bodies and five osteophytes (spondylophytes) were collected and prepared for microindentation test. In particular, indentations were performed on bone structural units of the cortical shell (along axial, circumferential and radial directions), of the endplates (along the anterio-posterior and lateral directions), of the trabecular bone (along the axial and transverse directions) and of the spondylophytes (along the axial direction). A total of 3164 indentations down to a maximum depth of 2.5 µm were performed and the indentation modulus was computed for each measurement. RESULTS: The cortical shell showed an orthotropic behavior (indentation modulus, Ei, higher if measured along the axial direction, 14.6±2.8 GPa, compared to the circumferential one, 12.3±3.5 GPa, and radial one, 8.3±3.1 GPa). Moreover, the cortical endplates (similar Ei along the antero-posterior, 13.0±2.9 GPa, and along the lateral, 12.0±3.0 GPa, directions) and the trabecular bone (Ei= 13.7±3.4 GPa along the axial direction versus Ei=10.9±3.7 GPa along the transverse one) showed transversal isotropy behavior. Furthermore, the spondylophytes showed the lower mechanical properties measured along the axial direction (Ei=10.5±3.3 GPa). CONCLUSIONS: The original results presented in this study improve our understanding of vertebral biomechanics and can be helpful to define the material properties of the vertebral substructures in computational models such as FE analysis.

Poly(ADP-ribose) polymerase family member 14 (PARP14) is a novel effector of the JNK2-dependent pro-survival signal in multiple myeloma.

Regulation of cell survival is a key part of the pathogenesis of multiple myeloma (MM). Jun N-terminal kinase (JNK) signaling has been implicated in MM pathogenesis, but its function is unclear. To elucidate the role of JNK in MM, we evaluated the specific functions of the two major JNK proteins, JNK1 and JNK2. We show here that JNK2 is constitutively activated in a panel of MM cell lines and primary tumors. Using loss-of-function studies, we demonstrate that JNK2 is required for the survival of myeloma cells and constitutively suppresses JNK1-mediated apoptosis by affecting expression of poly(ADP-ribose) polymerase (PARP)14, a key regulator of B-cell survival. Strikingly, we found that PARP14 is highly expressed in myeloma plasma cells and associated with disease progression and poor survival. Overexpression of PARP14 completely rescued myeloma cells from apoptosis induced by JNK2 knockdown, indicating that PARP14 is critically involved in JNK2-dependent survival. Mechanistically, PARP14 was found to promote the survival of myeloma cells by binding and inhibiting JNK1. Moreover, inhibition of PARP14 enhances the sensitization of MM cells to anti-myeloma agents. Our findings reveal a novel regulatory pathway in myeloma cells through which JNK2 signals cell survival via PARP14, and identify PARP14 as a potential therapeutic target in myeloma.

Finite element analysis of cancellous bone failure in the vertebral body of healthy and osteoporotic subjects.

The aim of this work is to assess the fracture risk prediction of the cancellous bone in the body of a lumbar vertebra when the mechanical parameters of the bone, i.e. stiffness, porosity, and strength anisotropy, of elderly and osteoporotic subjects are considered. For this purpose, a non-linear three-dimensional continuum-based finite element model of the lumbar functional spinal unit L4-L5 was created and strength analyses of the spongy tissue of the vertebral body were carried out. A fabric-dependent strength criterion, which accounts for the micro-architecture of the cancellous bone, based on histomorphometric analyses was used. The strength analyses have shown that the cancellous bone of none of the subject types undergoes failure under loading applied during normal daily life like axial compression; however, bone failure occurs for the osteoporotic segment, subjected to a combination of the compression preloading and moments in the sagittal or in the frontal plane, which are conditions that may not be considered to occur 'daily'. In particular, critical stress conditions are met because of the high porosity values in the horizontal direction within the cancellous bone. The computational approach presented in the paper can potentially predict the material fracture risk of the cancellous bone in the vertebral body and it may be usefully employed to draw failure maps representing, for a given micro-architecture of the spongy tissue, the critical loading conditions (forces and moments) that may lead to such a risk. This approach could be further developed in order to assess the effectiveness of biomedical devices within an engineering approach to the clinical problem of the spinal diseases.

Role of the JNK pathway in NMDA-mediated excitotoxicity of cortical neurons.

Excitotoxic insults induce c-Jun N-terminal kinase (JNK) activation, which leads to neuronal death and contributes to many neurological conditions such as cerebral ischemia and neurodegenerative disorders. The action of JNK can be inhibited by the D-retro-inverso form of JNK inhibitor peptide (D-JNKI1), which totally prevents death induced by N-methyl-D-aspartate (NMDA) in vitro and strongly protects against different in vivo paradigms of excitotoxicity. To obtain optimal neuroprotection, it is imperative to elucidate the prosurvival action of D-JNKI1 and the death pathways that it inhibits. In cortical neuronal cultures, we first investigate the pathways by which NMDA induces JNK activation and show a rapid and selective phosphorylation of mitogen-activated protein kinase kinase 7 (MKK7), whereas the only other known JNK activator, mitogen-activated protein kinase kinase 4 (MKK4), was unaffected. We then analyze the action of D-JNKI1 on four JNK targets containing a JNK-binding domain: MAPK-activating death domain-containing protein/differentially expressed in normal and neoplastic cells (MADD/DENN), MKK7, MKK4 and JNK-interacting protein-1 (IB1/JIP-1).

Mutual cross-talk between reactive oxygen species and nuclear factor-kappa B: molecular basis and biological significance.

Reactive oxygen species (ROS) are emerging as key effectors in signal transduction. This role of ROS is especially evident in the pathways leading to programmed cell death (PCD) elicited in response to certain stress stimuli and cytokines. In these pathways, cytotoxic ROS signaling appears to be mediated in part by activation of the c-Jun-N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) cascade. Another pathway that is under ROS-mediated control in some systems is that leading to activation of transcription factor nuclear factor-kappa B (NF-kappaB), which is a central regulator of immunity, inflammation and cell survival. Remarkably, new evidence has unveiled the existence of a reciprocal, negative control that NF-kappaB exerts on ROS and JNK activities. This NF-kappaB-imposed restraint on ROS and JNK signaling is crucial for antagonism of PCD elicited by the proinflammatory cytokine tumor necrosis factor (TNF)alpha and likely other triggers. Effectors of this antagonistic cross-talk between NF-kappaB and ROS/JNK pathways have recently been identified. Because of the key roles that the prosurvival function of NF-kappaB plays in organismal physiology and disease, gaining a further mechanistic understanding of this cross-talk and NF-kappaB-dependent survival may be key to developing new therapies for the treatment of widespread human illnesses, such as cancer and chronic inflammatory conditions.

Growth arrest- and DNA-damage-inducible 45beta gene inhibits c-Jun N-terminal kinase and extracellular signal-regulated kinase and decreases IL-1beta-induced apoptosis in insulin-producing INS-1E cells.

AIMS/HYPOTHESIS: IL-1beta is a candidate mediator of apoptotic beta cell destruction, a process that leads to type 1 diabetes and progression of type 2 diabetes. IL-1beta activates beta cell c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and p38, all of which are members of the mitogen-activated protein kinase (MAPK) family. Inhibition of JNK prevents IL-1beta-mediated beta cell destruction. In mouse embryo fibroblasts and 3DO T cells, overexpression of the gene encoding growth arrest and DNA-damage-inducible 45beta (Gadd45b) downregulates pro-apoptotic JNK signalling. The aim of this study was to investigate if Gadd45b prevents IL-1beta-induced beta cell MAPK signalling and apoptosis. MATERIALS: Rat insulinoma INS-1E cells and mouse beta-TC3 cells stably expressing Gadd45b were generated. The effects of Gadd45b expression on signalling by JNK, ERK and p38 were assessed by Western blotting and kinase assays. Apoptosis rate was measured by terminal deoxynucleotidyl-mediated dUTP-biotin nick end-labelling (TUNEL) and an ELISA designed to detect apoptotic nucleosomes. Expression of endogenous Gadd45b mRNA was measured by RT-PCR. RESULTS: In INS-1E and beta-TC3 cells, expression of Gadd45b inhibited IL-1beta-induced activation of JNK and ERK, but augmented IL-1beta-mediated p38 activity. IL-1beta-induced nitric oxide production and decreases in insulin content and secretion were reduced by GADD45beta. IL-1beta-induced apoptosis was reduced by GADD45beta by up to 77%. Although IL-1beta stimulated the time-dependent induction of endogenous Gadd45b in INS-1E cells and rat islets, expression levels were lower in these cells than in IL-1beta-exposed NIH-3T3 and 3DO T cells. CONCLUSIONS/INTERPRETATION: Inadequate induction of Gadd45b, which encodes a novel beta cell JNK and ERK inhibitor, may in part explain the pro-apoptotic response of beta cells to IL-1beta.

The NF-kappaB-mediated control of the JNK cascade in the antagonism of programmed cell death in health and disease.

NF-kappaB/Rel transcription factors have recently emerged as crucial regulators of cell survival. Activation of NF-kappaB antagonizes programmed cell death (PCD) induced by tumor necrosis factor-receptors (TNF-Rs) and several other triggers. This prosurvival activity of NF-kappaB participates in a wide range of biological processes, including immunity, lymphopoiesis and development. It is also crucial for pathogenesis of various cancers, chronic inflammation and certain hereditary disorders. This participation of NF-kappaB in survival signaling often involves an antagonism of PCD triggered by TNF-R-family receptors, and is mediated through a suppression of the formation of reactive oxygen species (ROS) and a control of sustained activation of the Jun-N-terminal kinase (JNK) cascade. Effectors of this antagonistic activity of NF-kappaB on this ROS/JNK pathway have been recently identified. Indeed, further delineating the mechanisms by which NF-kappaB promotes cell survival might hold the key to developing new highly effective therapies for treatment of widespread human diseases.

The NF-kappaB-mediated control of ROS and JNK signaling.

NF-kappaB/Rel transcription factors are best known for their roles in innate and adaptive immunity and inflammation. They also play a central role in promoting cell survival. This latter activity of NF-kappaB antagonizes programmed cell death (PCD) induced by the proinflammatory cytokine tumor necrosis factor (TNF)alpha and plays an important role in immunity, lymphopoiesis, osteogenesis, tumorigenesis and radio- and chemoresistance in cancer. With regard to TNFalpha, the NF-kappaB-mediated inhibition of PCD seems to involve an attenuation of the c-Jun-N-terminal kinase (JNK) cascade mediated through the induction of select downstream targets such as the caspase inhibitor XIAP, the zinc-finger protein A20, and the inhibitor of the MKK7/JNKK2 kinase, Gadd45beta/Myd118. Notably, NF-kappaB also blunts accumulation of reactive oxygen species (ROS), which themselves are pivotal elements for induction of PCD by TNFalpha, and this suppression of ROS formation mediates an additional protective activity recently ascribed to NF-kappaB. The antioxidant activity of NF-kappaB has been shown to depend upon upregulation of both Ferritin heavy chain (FHC)--a component of Ferritin, the primary iron-storage protein complex found in cells--and of the mitochondrial enzyme Mn++ superoxide dismutase (Mn-SOD). Indeed, the inductions of Mn-SOD and FHC represent another important means through which NF-kappaB controls proapoptotic JNK signaling triggered by TNFalpha. These findings might enable the development of new, more targeted approaches to treatment of diseases sustained by a deregulated activity of NF-kappaB, including some cancers and chronic inflammatory conditions.

c-FLIP efficiently rescues TRAF-2-/- cells from TNF-induced apoptosis.

For the past 20 years, it has been known that preparations of Tumor Necrosis Factor alpha (TNF) fail to induce apoptosis due to cytoprotective responses that render cells resistant to its cytotoxic activity. Here we show that TRAF-2-/- embryonic fibroblasts express reduced levels of the anti-apoptotic molecule c-FLIP due to extensive degradation of the protein. Reconstitution of TRAF-2-/- EF with c-FLIP is sufficient for resistance to TNF toxicity. Our results strengthen the role of c-FLIP in protecting cells from the cytotoxic effect of TNF and have implication for the treatment of inflammatory and proliferative disorders.

Induction of gadd45beta by NF-kappaB downregulates pro-apoptotic JNK signalling.

In addition to coordinating immune and inflammatory responses, NF-kappaB/Rel transcription factors control cell survival. Normally, NF-kappaB dimers are sequestered in the cytoplasm by binding to inhibitory IkappaB proteins, and can be activated rapidly by signals that induce the sequential phosphorylation and proteolysis of IkappaBs. Activation of NF-kappaB antagonizes apoptosis or programmed cell death by numerous triggers, including the ligand engagement of 'death receptors' such as tumour-necrosis factor (TNF) receptor. The anti-apoptotic activity of NF-kappaB is also crucial to oncogenesis and to chemo- and radio-resistance in cancer. Cytoprotection by NF-kappaB involves the activation of pro-survival genes; however, its basis remains poorly understood. Here we report that NF-kappaB complexes downregulate the c-Jun amino-terminal kinase (JNK) cascade, thus establishing a link between the NF-kappaB and the JNK pathways. This link involves the transcriptional upregulation of gadd45beta/myd118 (ref. 4), which downregulates JNK signalling induced by the TNF receptor (TNF-R). This NF-kappaB-dependent inhibition of the JNK pathway is central to the control of cell death. Our findings define a protective mechanism that is mediated by NF-kappaB complexes and establish a role for the persistent activation of JNK in the apoptotic response to TNF-alpha.

Physical and functional interaction of filamin (actin-binding protein-280) and tumor necrosis factor receptor-associated factor 2.

Tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2) is an intracellular protein involved in signal transduction from TNF receptor I and II and related receptors. TRAF2 is required for TNF-induced activation of c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), and TRAF2 can also mediate activation of NF-kappaB. Here we have identified the actin-binding protein Filamin (actin-binding protein-280) as a TRAF2-interacting protein. Filamin binds to the Ring zinc finger domain of TRAF2. Overexpressed Filamin inhibits TRAF2-induced activation of JNK/SAPK and of NF-kappaB. Furthermore, ectopically expressed Filamin inhibits NF-kappaB activation induced via TNF, interleukin-1, Toll receptors, and TRAF6 but not activation induced via overexpression of NIK, a downstream effector in these pathways. Importantly, TNF fails to activate SAPK or NF-kappaB in a human melanoma cell line deficient in Filamin. Reintroduction of Filamin into these cells restores the TNF response. The data imply a role for Filamin in inflammatory signal transduction pathways.

Required and nonessential functions of nuclear factor-kappa B in bone cells.

Nuclear factor-kappa B (NF-kappaB) is a set of five polypeptide transcription factors, called p50, p52, p65 (also called Rel A), Rel B, and c-Rel, which regulate the expression of a variety of genes involved in immune and inflammatory responses. They were originally named because they were considered essential regulators of B cell kappa light chain expression. More recent studies indicate that NF-kappaB proteins are involved in the regulation of a variety of other cell functions, including cell proliferation, responses to stress, and apoptosis. NF-kappaB heterodimers reside in the cytoplasm of cells bound to inhibitory proteins, the two commonest of which are IkappaBalpha and IkappaBbeta, which prevent NF-kappaB from entering the nucleus. When cells are stimulated, IkappaB is phosphorylated by specific IkappaB kinases and subsequently is ubiquitinated and degraded in proteosomes. This allows NF-kappaB to translocate to the nucleus to regulate the expression of a growing list of genes, including the proinflammatory cytokines, interleukin-1 (IL-1), IL-6, and tumor necrosis factor. IL-1 and tumor necrosis factor in turn also regulate the expression of NF-kappaB. Thus, once activated, NF-kappaB may be involved in upregulatory loops, which can amplify the effects of the initiating stimulus. Because these proinflammatory cytokines have been implicated in the pathogenesis of estrogen deficiency and inflammation-related bone loss, it is likely that NF-kappaB has a significant role in the increased generation and function of osteoclasts in these circumstances. However, an unexpected and essential role of NF-kappaB in the formation of osteoclasts during development was discovered recently after the generation of knockout mice, which lack the expression of the p50 and p52 subunits. This paper will describe recent studies that reveal an essential role for NF-kappaB signaling in the generation of osteoclasts and that suggest that NF-kappaB may also play a key central role in the activation and survival of osteoclasts in conditions in which osteoclastogenesis is upregulated.

Evidence for distinct intracellular signaling pathways in CD34+ progenitor to dendritic cell differentiation from a human cell line model.

Intracellular signals that mediate differentiation of pluripotent hemopoietic progenitors to dendritic cells (DC) are largely undefined. We have previously shown that protein kinase C (PKC) activation (with phorbol ester (PMA) alone) specifically induces differentiation of primary human CD34+ hemopoietic progenitor cells (HPC) to mature DC. We now find that cytokine-driven (granulocyte-macrophage CSF and TNF-alpha) CD34+ HPC-->DC differentiation is preferentially blocked by inhibitors of PKC activation. To further identify intracellular signals and downstream events important in CD34+ HPC-->DC differentiation we have characterized a human leukemic cell line model of this process. The CD34+ myelomonocytic cell line KG1 differentiates into dendritic-like cells in response to granulocyte-macrophage CSF plus TNF-alpha, or PMA (with or without the calcium ionophore ionomycin, or TNF-alpha), with different stimuli mediating different aspects of the process. Phenotypic DC characteristics of KG1 dendritic-like cells include morphology (loosely adherent cells with long neurite processes), MHC I+/MHC IIbright/CD83+/CD86+/CD14- surface Ag expression, and RelB and DC-CK1 gene expression. Functional DC characteristics include fluid phase macromolecule uptake (FITC-dextran) and activation of resting T cells. Comparison of KG1 to the PMA-unresponsive subline KG1a reveals differences in expression of TNF receptors 1 and 2; PKC isoforms alpha, beta I, beta II, and mu; and RelB, suggesting that these components/pathways are important for DC differentiation. Together, these findings demonstrate that cytokine or phorbol ester stimulation of KG1 is a model of human CD34+ HPC to DC differentiation and suggest that specific intracellular signaling pathways mediate specific events in DC lineage commitment.

IkappaB-alpha enhances transactivation by the HOXB7 homeodomain-containing protein.

Combinatorial interactions between distinct transcription factors generate specificity in the controlled expression of target genes. In this report, we demonstrated that the HOXB7 homeodomain-containing protein, which plays a key role in development and differentiation, physically interacted in vitro with IkappaB-alpha, an inhibitor of NF-kappaB activity. This interaction was mediated by the IkappaB-alpha ankyrin repeats and C-terminal domain as well as by the HOXB7 N-terminal domain. In transient transfection experiments, IkappaB-alpha markedly increased HOXB7-dependent transcription from a reporter plasmid containing a homeodomain consensus-binding sequence. This report therefore showed a novel function for IkappaB-alpha, namely a positive regulation of transcriptional activation by homeodomain-containing proteins.

Mice deficient in nuclear factor (NF)-kappa B/p52 present with defects in humoral responses, germinal center reactions, and splenic microarchitecture.

p52 is a subunit of nuclear factor (NF)-kappa B transcription factors, most closely related to p50. Previously, we have shown that p52, but not p50 homodimers can form transactivating complexes when associated with Bcl-3, an unusual member of the I kappa B family. To determine nonredundant physiologic roles of p52, we generated mice deficient in p52. Null mutant mice were impaired in their ability to generate antibodies to T-dependent antigens, consistent with an absence of B cell follicles and follicular dendritic cell networks in secondary lymphoid organs, and an inability to form germinal centers. Furthermore, the splenic marginal zone was disrupted. These phenotypes are largely overlapping with those observed in Bcl-3 knockout animals, but distinct from those of p50 knockouts, supporting the notion of a physiologically relevant complex of p52 homodimers and Bcl-3. Adoptive transfer experiments further suggest that such a complex may be critical in accessory cell functions during antigen-specific immune reactions. Possible roles of p52 and Bcl-3 are discussed that may underlie the oncogenic potential of these proteins, as evidenced by recurrent chromosomal translocations of their genes in lymphoid tumors.

The signal response of IkappaB alpha is regulated by transferable N- and C-terminal domains.

IkappaB alpha retains the transcription factor NF-kappaB in the cytoplasm, thus inhibiting its function. Various stimuli inactivate IkappaB alpha by triggering phosphorylation of the N-terminal residues Ser32 and Ser36. Phosphorylation of both serines is demonstrated directly by phosphopeptide mapping utilizing calpain protease, which cuts approximately 60 residues from the N terminus, and by analysis of mutants lacking one or both serine residues. Phosphorylation is followed by rapid proteolysis, and the liberated NF-kappaB translocates to the nucleus, where it activates transcription of its target genes. Transfer of the N-terminal domain of IkappaB alpha to the ankyrin domain of the related oncoprotein Bcl-3 or to the unrelated protein glutathione S-transferase confers signal-induced phosphorylation on the resulting chimeric proteins. If the C-terminal domain of IkappaB alpha is transferred as well, the resulting chimeras exhibit both signal-induced phosphorylation and rapid proteolysis. Thus, the signal response of IkappaB alpha is controlled by transferable N-terminal and C-terminal domains.

Critical roles for the Bcl-3 oncoprotein in T cell-mediated immunity, splenic microarchitecture, and germinal center reactions.

Chromosomal translocations of bcl-3 are associated with chronic B cell lymphocytic leukemias. Previously, we have shown that Bcl-3, a distinct member of the I kappa B family, may function as a positive regulator of NF-kappa B activity, although its physiologic roles remained unknown. To uncover these roles, we generated Bcl-3-deficient mice. Mutant mice, but not their littermate controls, succumb to T. gondii owing to failure to mount a protective T helper 1 immune response. Bcl-3-deficient mice are also impaired in germinal center reactions and T-dependent antibody responses to influenza virus. The results reveal critical roles for Bcl-3 in antigen-specific priming of T and B cells. Altered microarchitecture of secondary lymphoid organs in mutant mice, including partial loss of B cells, may underlie the immunologic defects. The implied role of Bcl-3 in maintaining B cells in wild-type mice may related to its oncogenic potential.