Ronald D. Graff

NCAM stimulates the ras‐MAPK pathway and CREB phosphorylation in neuronal cells

The neural cell adhesion molecule NCAM plays an important role in axonal growth, learning, and memory. A signaling pathway has been elucidated in which clustering of the NCAM140 isoform in the neural plasma membrane stimulated the activating phosphorylation of mitogen-activated protein kinases (MAPKs) and the transcription factor cyclic AMP response-element binding protein (CREB). NCAM clustering transiently induced dual phosphorylation (activation) of the MAPKs ERK1 and ERK2 (extracellular signal-regulated kinases) by a pathway regulated by the focal adhesion kinase p125fak, p59fyn, Ras, and MAPK kinase. CREB phosphorylation at serine 133 induced by NCAM was dependent in part on an intact MAPK pathway. c-Jun N-terminal kinase, which is associated with apoptosis and cellular stress, was not activated by NCAM. Inhibition of the MAPK pathway in rat cerebellar neuron cultures selectively reduced NCAM-stimulated neurite outgrowth. These results define an NCAM signal transduction mechanism with the potential for modulating the expression of genes needed for axonal growth, survival, and synaptic plasticity.

ATP release by mechanically loaded porcine chondrons in pellet culture

OBJECTIVE To determine whether ATP is released from chondrocytes during mechanical stimulation and whether degradation of ATP generates inorganic pyrophosphate in chondron pellet cultures. METHODS Chondron pellets were formed from 1.6 x 10(6) cells that had been enzymatically isolated from porcine articular cartilage. ATP was measured in media from cultures at rest and during fluid movement and cyclic compression. ATP hydrolysis was examined by high-performance liquid chromatography following the addition of gamma32P-ATP to resting cultures. RESULTS Pellet cultures at rest maintained a steady-state concentration of 2-4 nM ATP in 2 ml of medium. The ATP concentration increased 5-12-fold with cyclic compression (7.5 and 15 kPa at 0.5 Hz), then decreased to preloading levels within 60 minutes despite continued loading. A subsequent increase in pressure stimulated a further increase in ATP release, suggesting that chondrocytes desensitize to load. Cell viability was similar for pellets at rest and up to 24 hours after compression. ATP released in response to mechanical stimulation was inhibited 50% by 0.5 mM octanol, suggesting a regulated mechanism for ATP release. Exogenous ATP was rapidly hydrolyzed to pyrophosphate in resting cultures. CONCLUSION The occurrence of basal levels of extracellular ATP in the presence of pyrophosphohydrolase activity indicates that ATP was continuously released by chondrocytes at rest. Considering that chondrocytes express purinoceptors that respond to ATP, we suggest a role for ATP in extracellular signaling by chondrocytes in response to mechanical load. ATP released by chondrocytes in response to mechanical load is a likely source of pyrophosphate in calcium pyrophosphate dihydrate crystal deposition diseases.

Tissue transglutaminase localization and activity regulation in the extracellular matrix of articular cartilage.

Tissue transglutaminase (tTG) catalyzes a Ca2+‐dependent transglutaminase (TGase) activity which cross‐links proteins and stabilizes many tissues [C.S. Greenberg et al. FASEB J. 5 (1991) 3071]. Because cartilage is subjected to great stress in vivo, an enzyme that strengthens and stabilizes tissue could play an integral role in maintaining cartilage integrity. The purpose of this study was to determine if active tTG is present in the extracellular matrix (ECM) of adult human osteoarthritic articular cartilage. Using a TGase activity assay along with immunolabeling for tTG of cartilage sections, TGase activity and tTG immunoreactivity were localized in the ECM in cartilage sections, predominantly in the superficial layer. Previous in vitro studies have demonstrated that the Mg‐GTP complex inhibits the TGase activity of tTG [T.S. Lai et al. J. Biol. Chem. 273 (1998) 1776]. To investigate the in situ regulation of the TGase activity of tTG, a TGase activity assay was done with a dose response of GTP, measuring incorporation of fluorescein cadaverine. TGase activity was inhibited by GTP in a similar manner as in vitro. These results not only confirm tTG presence in the ECM, but also indicate tTG as the major TGase activity of the ECM. Secondly, the study provides a possible mechanism by which extracellular tTG is regulated in vivo.

Annulus cells release ATP in response to vibratory loading in vitro

Mechanical forces regulate the developmental path and phenotype of a variety of tissues and cultured cells. Vibratory loading as a mechanical stimulus occurs in connective tissues due to energy returned from ground reaction forces, as well as a mechanical input from use of motorized tools and vehicles. Structures in the spine may be particularly at risk when exposed to destructive vibratory stimuli. Cells from many tissues respond to mechanical stimuli, such as fluid flow, by increasing intracellular calcium concentration ([Ca2+]ic) and releasing adenosine 5′‐triphosphate (ATP), extracellularly, as a mediator to activate signaling pathways. Therefore, we examined whether ATP is released from rabbit (rAN) and human (hAN) intervertebral disc annulus cells in response to vibratory loading. ATP release from annulus cells by vibratory stimulation as well as in control cells was quantitated using a firefly luciferin‐luciferase assay. Cultured hAN and rAN cells had a basal level of extracellular ATP ([ATP]ec) in the range of 1–1.5 nM. Vibratory loading of hAN cells stimulated ATP release, reaching a net maximum [ATP] within 10 min of continuous vibration, and shortly thereafter, [ATP] declined and returned to below baseline level. [ATP] in the supernatant fluid of hAN cells was significantly reduced compared to the control level when the cells received vibration for longer than 15 min. In rAN cells, [ATP] was increased in response to vibratory loading, attaining a level significantly greater than that of the control after 30 min of continuous vibration. Results of the current study show that resting annulus cells secrete ATP and maintain a basal [ATP]ec. Annulus cells may use this nucleotide as a signaling messenger in an autocrine/paracrine fashion in response to vibratory loading. Rapid degradation of ATP to ADP may alternatively modulate cellular responses. It is hypothesized that exposure to repetitive, complex vibration regimens may activate signaling pathways that regulate matrix destruction in the disc. As in tendon cells, ATP may block subsequent responses to load and modulate the vibration response. Rabbit annulus cells were used as a readily obtainable source of cells in development of an animal model for testing effects of vibration on the disc. Human cells obtained from discarded surgical specimens were used to correlate responses of animal to human cells.

Extracellular nucleotides, cartilage stress, and calcium crystal formation.

&NA; Nucleotides are released by chondrocytes at rest and in response to mechanical stimulation. Extracellular nucleotides are metabolized by a variety of ectoenzymes, producing free phosphate (Pi) or pyrophosphate (PPi) and promoting matrix mineralization. Ectoenzymes are differentially localized in cartilage and may be co‐released with nucleotides during mechanical stimulation. Extracellular nucleotides can also serve as substrates and/or modulators of enzymes such as tissue transglutaminase and ecto‐protein kinases that modify matrix proteins and regulate crystal deposition or growth. Understanding the evolution of osteoarthritis and calcium crystal deposition diseases will require clearer knowledge of the functions of nucleotides and ectoenzymes in the cartilage extracellular matrix.

Development of selective tolerance to interleukin-1β by human chondrocytes in vitro

Interleukin‐1 induces release of NO and PGE2 and production of matrix degrading enzymes in chondrocytes. In osteoarthritis (OA), IL‐1 continually, or episodically, acts on chondrocytes in a paracrine and autocrine manner. Human chondrocytes in chondron pellet culture were treated chronically (up to 14 days) with IL‐1β. Chondrons from OA articular cartilage were cultured for 3 weeks before treatment with IL‐1β (0.05–10 ng/ml) for an additional 2 weeks. Spontaneous release of NO and IL‐1β declined over the pretreatment period. In response to IL‐1β (0.1 ng/ml), NO and PGE2 release was maximal on Day 2 or 3 and then declined to near basal level by Day 14. Synthesis was recovered by addition of 1 ng/ml IL‐1β on Day 11. Expression of inducible nitric oxide synthase (iNOS), detected by immunofluorescence, was elevated on Day 2 and declined through Day 14, which coordinated with the pattern of NO release. On the other hand, IL‐1β‐induced MMP‐13 synthesis was elevated on Day 3, declined on Day 5, and then increased again through Day 14. IL‐1β increased glucose consumption and lactate production throughout the treatment. IL‐1β stimulated proteoglycan degradation in the early days and inhibited proteoglycan synthesis through Day 14. Chondron pellet cultures from non‐OA cartilage released the same amount of NO but produced less PGE2 and MMP‐13 in response to IL‐1β than OA cultures. Like the OA, IL‐1β‐induced NO and PGE2 release decreased over time. In conclusion, with prolonged exposure to IL‐1β, human chondrocytes develop selective tolerance involving NO and PGE2 release but not MMP‐13 production, metabolic activity, or matrix metabolism.

An evaluation of carrier agents for desferoxamine, an up-regulator of vascular endothelial growth factor

Avascularity and hypoxia result in avascular necrosis and play a negative role in fracture healing. The FDA-approved iron chelating agent, desferoxamine (DFO) in a liquid form, has been shown to induce angiogenesis and improve fracture healing through upregulation of the vascular endothelial growth factor. We were concerned that local injection of DFO would either fail to adequately deliver sufficient drug to the desired site or lead to undesired delivery to adjacent sites. Therefore, a sustained release delivery system was desirable to direct DFO to the intended site. Calcium sulfate pellets, collagen sponges, and demineralized cortical bone matrix were all evaluated as potentially controlled release systems for DFO using a fetal mouse metatarsal angiogenesis assay. Angiogenesis was analyzed using a vascularity grading scale, by measuring the mean vessel length of the 5 longest vessels, and by counting the mean number of vessels per metatarsal. Although there was some evidence of angiogenesis with all three carriers, DFO loaded CaSO4 pellets increased vascularity grading, the mean length of the five longest vessels, and the mean number of vessels, all by statistically significant margins versus the control. These results suggest that CaSO4 pellets could be used as a viable, nontoxic, controlled release system for DFO in clinical situations where increased angiogenesis and bone growth are desirable.

Microtubule dynamics in a cytosolic extract of fetal rat brain.

Brain microtubule dynamics were studied by video-enhanced differential interference contrast microscopy in a cytosolic extract from fetal rat brain, prepared under conditions designed to produce minimal alterations in microtubule stability. With urchin sperm axoneme fragments as nucleation seeds, the extract was shown to support cellular-like microtubule dynamics. Microtubules elongated from one end of the axonemes, and did not spontaneously self-assemble in the absence of axonemes. The following microtubule kinetic parameters were measured in the extract: velocity of elongation (8.1 mm/min), velocity of rapid shortening (5.8 mm/min), catastrophe frequency (0.17 min-1), and rescue frequency (1 min-1). These parameters were in close agreement with reported values for growth cones of living neurons. Microtubule properties in the fetal brain extract were shown to be affected by agents with known effects on the cytoskeleton. pp60c-src, a tyrosine kinase important in cell adhesion molecule-dependent axon growth, caused small increases in the frequency of microtubule catastrophe (0.31 min-1) and rescue (2 min-1) without changing the velocities of elongation or rapid-shortening. Although pp60c-src phosphorylated purified porcine brain tubulin in vitro, it did not elicit significant changes in its polymerization properties, suggesting that other cytoskeletal proteins in the brain extract are involved in modulating microtubule dynamics. The cytosolic extract of fetal rat brain provides a useful system for studying regulation of microtubule assembly in neuronal growth cones.

The effect of methylprednisolone intravenous infusion on the expression of ciliary neurotrophic factor in a rat spinal cord injury model

BACKGROUND CONTEXT Methylprednisolone (MP) infusion after acute spinal cord injury (SCI) remains controversial despite large randomized studies, including the National Acute Spinal Cord Injury Studies (NASCIS). PURPOSE To determine the effect of NASCIS protocol MP infusion on the expression of ciliary neurotrophic factor (CNTF), a neuroprotective cytokine, in a rat model after SCI. STUDY DESIGN Animal laboratory study. METHODS Thirty rats were randomized into an MP infusion group (intravenous [IV]-MP) versus normal saline (NS) control group (IV-NS) after a standardized SCI. Ciliary neurotrophic factor expression was measured by reverse transcription-polymerase chain reaction at 6, 12, 24, 48, and 72 hours post-SCI. RESULTS Mean CNTF expression was diminished in the MP group at 12 (p=.006) and 24 (p=.008) hours postinjury compared with the control group. Expression of CNTF was not significantly different between the groups at 6, 48, and 72 hours post-SCI. CONCLUSIONS Standardized MP infusion post-SCI reduces CNTF activation in a rat SCI model. Further study is needed to determine if this effect is seen in human SCIs.