The induction of programmed cell death, or apoptosis, involves activation of a signalling system, many elements of which remain unknown. The sphingomyelin pathway, initiated by hydrolysis of the phospholipid sphingomyelin in the cell membrane to generate the second messenger ceramide, is thought to mediate apoptosis in response to tumour-necrosis factor (TNF)-alpha, to Fas ligand and to X-rays. It is not known whether it plays a role in the stimulation of other forms of stress-induced apoptosis. Given that environmental stresses also stimulate a stress-activated protein kinase (SAPK/JNK), the sphingomyelin and SAPK/JNK signalling systems may be coordinated in induction of apoptosis. Here we report that ceramide initiates apoptosis through the SAPK cascade and provide evidence for a signalling mechanism that integrates cytokine- and stress-activated apoptosis.

Objective. Prior reports document macrophage and lymphocyte infiltration with proinflammatory cytokine expression in pathologic intervertebral disc (IVD) tissues. Nevertheless, the role of the Th17 lymphocyte lineage in mediating disc disease remains uninvestigated. We undertook this study to evaluate the immunophenotype of pathologic IVD specimens, including interleukin-17 (IL-17) expression, from surgically obtained IVD tissue and from nondegenerated autopsy control tissue.Methods. Surgical IVD tissues were procured from patients with degenerative disc disease (n ‫؍‬ 25) or herniated IVDs (n ‫؍‬ 12); nondegenerated autopsy control tissue was also obtained (n ‫؍‬ 8) from the anulus fibrosus and nucleus pulposus regions. Immunohistochemistry was performed for cell surface antigens (CD68 for macrophages, CD4 for lymphocytes) and various cytokines, with differences in cellularity and target immunoreactivity scores analyzed between surgical tissue groups and between autopsy control tissue regions.Results. Immunoreactivity for IL-4, IL-6, IL-12, and interferon-␥ (IFN␥) was modest in surgical IVD tissue, although expression was higher in herniated IVD samples and virtually nonexistent in control samples. The Th17 lymphocyte product IL-17 was present in >70% of surgical tissue fields, and among control samples was detected rarely in anulus fibrosus regions and modestly in nucleus pulposus regions. Macrophages were prevalent in surgical tissues, particularly herniated IVD samples, and lymphocytes were expectedly scarce. Control tissue revealed lesser infiltration by macrophages and a near absence of lymphocytes.Conclusion. Greater IFN␥ positivity, macrophage presence, and cellularity in herniated IVDs suggests a pattern of Th1 lymphocyte activation in this pathology. Remarkable pathologic IVD tissue expression of IL-17 is a novel finding that contrasts markedly with low levels of IL-17 in autopsy control tissue. These findings suggest involvement of Th17 lymphocytes in the pathomechanism of disc degeneration.

The potential involvement of ceramiderelated signaling processes in the induction of apoptosis by tumor necrosis factor a was assessed by multiple biochemical strategies in the human leukemic cell lines HL-60 and U937 and the murine fibrosarcoma cell lines L929/LM and WEHI 164/13. Exposure of these cells to tumor necrosis factor a resulted in internucleosomal cleavage of genomic DNA, yielding laddered patterns of oligonucleosomal fragments characteristic of apoptosis when resolved by agarose gel electrophoresis; similar responses were observed after exposure to exogenous sphingomyelinase or synthetic ceramides. Quantitative spectrofluorophotometry demonstrated that these treatments promoted time-and concentration-dependent degradation of DNA, resulting in the formation ofand eventual release ofsmall DNA fragments (<3.0 kb). Corresponding damage to bulk DNA was demonstrated by enhanced-fluorescence alkaline unwinding analysis. DNA fragmentation was not induced by phospholipase C or synthetic diglyceride; in fact, the effects of sphingomyelinase and ceramide were substantially reduced by coexposure to these agents, suggesting opposing roles for diglyceride-and ceramide-mediated signals in the regulation of apoptosis. Phospholipase A2 and arachidonic acid failed to promote DNA fragmentation, as did phospholipase D. Characterization of DNA strand breaks by alkaline and neutral elution analyses confirmed that ceramide action was restricted to breakage of mature, double-stranded DNA but not of nascent DNA. The induction of DNA damage was associated with appearance of apoptotic morphology and decreased clonogenicity. These results demonstrate that the ceramidedependent signaling system selectively induces apoptosis and raise the possibility that ceramide-activated enzymes represent important components in a signaling cascade involved in the regulation of programmed cell death.Programmed cell death, or apoptosis, is an active, energydependent process through which living cells participate in their own destruction and is initiated by a variety of physiological and pharmacological stimuli (1-4). A fundamental component of this response is the stereotypical degradation of genomic DNA to oligonucleosomal fragments (3, 4). The inflammatory cytokine tumor necrosis factor a (TNF-a) has been shown to initiate apoptotic cell death and DNA fragmentation in several mammalian cell lines, including the human leukemia cell lines HL-60 (5, 6) and U937 (6-8) and the murine fibrosarcoma cell lines L929 (9, 10) and WEHI (11,12). Two subtypes of TNF receptor, a high-affinity, 75-kDa (type A) form and a low-affinity, 55-kDa (type B) form, are expressed in comparable copy numbers in U937 and HL-60 cells (13); activation of the latter species has beenThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. implicated in the induction of apoptotic DNA degradation and cell death in bot...

Determinants of di erentiation and apoptosis in myelomonocytic leukemia cells (U937) exposed to the novel hybrid polar compound SAHA (suberoylanilide hydroxamic acid) have been examined. In contrast to hexamethylenbisacetamide (HMBA), SAHA-related maturation was limited and accompanied by marked cytoxicity. SAHA-mediated apoptosis occurred within the G 0 G 1 and S phase populations, and was associated with decreased mitochondrial membrane potential, caspase-3 activation, PARP degradation, hypophosphorylation/cleavage of pRB, and down-regulation of c-Myc, c-Myb, and B-Myb. Enforced expression of Bcl-2 or Bclx L inhibited SAHA-induced apoptosis, but only modestly potentiated di erentiation. While SAHA induced the cyclin-dependent kinase inhibitor p21 CIP1 , antisense ablation of this CDKI increased, rather than decreased, SAHA-related lethality. In contrast, conditional expression of wild-type p53 failed to modify SAHA actions, but markedly potentiated HMBA-induced apoptosis. Finally, SAHA modestly increased expression/activation of the stress-activated protein kinase (SAPK/JNK); moreover, SAHA-related lethality was partially attenuated by a dominant-negative c-Jun mutant protein (TAM67). SAHA did not stimulate mitogen-activated protein kinase (MAPK), nor was lethality diminished by the speci®c MEK/MAPK inhibitor PD98059. These ®ndings indicate that SAHA potently induces apoptosis in human leukemia cells via a pathway that is p53-independent but at least partially regulated by Bcl-2/Bcl-x L , p21 CIP1 , and the c-Jun/AP-1 signaling cascade.

To study the mechanisms by which catecholamines regulate hepatocyte proliferation after partial hepatectomy (PHX), hepatocytes were isolated from adult male rats 24 h after sham operation or two-thirds PHX and treated with catecholamines and other agonists. In freshly isolated sham cells, p42 mitogen-activated protein (MAP) kinase activity was stimulated by the ␣ 1 -adrenergic agonist phenylephrine (PHE). Activation of p42 MAP kinase by growth factors was blunted by pretreatment of sham hepatocytes with glucagon but not by that with the ␤ 2 -adrenergic agonist isoproterenol (ISO). In PHX cells, the ability of PHE to activate p42 MAP kinase was dramatically reduced, whereas ISO became competent to inhibit p42 MAP kinase activation. PHE treatment of sham but not PHX and ISO treatment of PHX but not sham hepatocytes also activated the stress-activated protein (SAP) kinases p46/54 SAP kinase and p38 SAP kinase. These data demonstrate that an Partial hepatectomy (PHX) or acute dissociation and primary culturing of hepatocytes trigger their entry into the cell cycle, which is accompanied by alterations in the levels of expression of various liver-specific proteins (14, 34). Epidermal growth factor (EGF), hepatocyte growth factor, and insulin have been shown to induce DNA synthesis in cultured hepatocytes or in quiescent liver (14,29,34). Catecholamines have been shown to increase DNA synthesis in quiescent liver via stimulation of ␣ 1 -adrenergic receptors (ARs) (6,14,18,24,27,34,44,51). The role(s) of ␤ 2 -ARs in the control of hepatic DNA synthesis is less clear, as both stimulatory and inhibitory effects have been described elsewhere (13,15,35). Previous studies have demonstrated that PHX or primary culture of rat hepatocytes significantly increases ␤ 2 -AR, and decreases ␣ 1 -AR, expression and function (24,35,38). However, the capacity of this receptor switch to modulate many of the recently discovered signal transduction pathways, such as the Raf/mitogen-activated protein (MAP) kinase cascade, and its role in the regulation of hepatocyte regeneration are unclear.Raf-1 is a member of a family of serine/threonine protein kinases termed Raf-1, B-Raf, and A-Raf (41). Proto-oncogenes of the Raf family have been implicated in hepatic carcinogenesis (2,11,17,34,40,41). Raf family members function in a signaling cascade that extends from the plasma membrane, via tyrosine kinase (16) and serpentine (22, 36) receptors, to the low-molecular-weight GTP-binding protein Ras. GTP-Ras binds and translocates Raf-1 to the plasma membrane, which leads to the activation of Raf-1 by multiple mechanisms (12,16,30,36). Raf family members in turn activate MEK1/2, which in turn activates the p42/44 MAP kinases, whose activation leads to modulation of downstream transcription factor activities (30, 46). The MAP kinase pathway can be negatively regulated by agonists that elevate cyclic AMP and activate protein kinase A (PK-A), such as glucagon and ␤ 2 -AR agonists (8,28,32,45). Activation of PK-A has been shown to inhibit or delay the act...