Joseph D. Etlinger

Cellular Physiology of STAT3: Where’s the Cytoplasmic Monomer?

In the standard model of cytokine-induced signal transducer and activator of transcription (STAT) protein family signaling to the cell nucleus, it is assumed that STAT3 is recruited to the cytoplasmic side of the cell surface receptor complex from within a cytosolic monomer pool. By using Superose-6 gel-filtration chromatography, we have discovered that there is little monomeric STAT3 (91 kDa) in the cytosol of liver cells (human hepatoma Hep3B cell line and rat liver). The bulk of STAT3 (and STAT1, STAT5a, and -b) was present in the cytosol as high molecular mass complexes in two broad distributions in the size range 200–400 kDa (“statosome I”) and 1–2 MDa (“statosome II”). Upon treatment of Hep3B cells with interleukin-6 (IL-6) for 30 min (i) cytosolic tyrosine-phosphorylated STAT3 was found to be in complexes of size ranging from 200–400 kDa to 1–2 MDa; (ii) a small pool of monomeric STAT3 and tyrosine-phosphorylated STAT3 eluting at 80–100 kDa was observed, and (iii) most of the cytoplasmic DNA-binding competent STAT3 (the so-called SIF-A “homodimer”) co-eluted with catalase at 230 kDa. In order to identify the protein components of the 200–400-kDa statosome I cytosolic complexes, we used the novel technique of antibody-subtracted differential protein display using anti-STAT3 antibody. Eight polypeptides in the size range from 20 to 114 kDa co-shifted with STAT3; three of these (p60, p20a, and p20b) were co-shifted in an IL-6-dependent manner. In-gel tryptic fragmentation and mass spectroscopy identified the major IL-6-dependent STAT3-co-shifted p60 protein as the chaperone GRP58/ER-60/ERp57. Taken together, these data (i) emphasize the absence of a detectable STAT3 monomer pool in the cytosol of cytokine-free liver cells as posited by the standard model, and (ii) suggest an alternative model for STAT signaling in which STAT3 proteins function in the cytoplasm as heteromeric complexes with accessory scaffolding proteins, including the chaperone GRP58.

Clenbuterol reduces degeneration of exercised or aged dystrophic (mdx) muscle

Evidence of dystrophic muscle degeneration in the hind limb muscles of young (20‐week‐old) treadmill‐exercised or aged (87‐week‐old) sedentary mdx mice was greatly reduced by treatment with clenbuterol, a β2‐adrenoceptor agonist. Daily treadmill exercise for 10 weeks increased the size of regions within the mdx plantaris but not the soleus or gastrocnemius muscles, in which necrotic muscle fibers or the absence of fibers was observed. Clenbuterol reduced the size of these abnormal regions from 21% of total muscle cross‐sectional area to levels (4%) found in sedentary mdx mice. In addition, the muscles obtained from aged clenbuterol‐treated mdx or wild‐type mice did not display the extensive fibrosis or fiber loss observed in untreated mdx mice. These observations are consistent with a mechanism of dystrophic muscle degeneration caused by work load–induced injury that is cumulative with aging and is opposed by β2‐adrenoceptor activation. Optimization of β2‐agonist treatment of muscular dystrophy in mdx mice may lead to a useful therapeutic modality for human forms of the disease.

Clenbuterol, a β2-Adrenoceptor Agonist, Improves Locomotor and Histological Outcomes after Spinal Cord Contusion in Rats

An important goal of rehabilitation following spinal cord injury is recovery of locomotor function and muscular strength. In the present studies, we determined whether the beta(2)-agonist, clenbuterol, can improve recovery of locomotor function following spinal cord injury. A model of spinal cord injury was examined in which four graded levels of contusion injury were produced in rats at the level of T10 with a weight-drop device. Locomotor recovery was determined with the Basso, Beattie, and Bresnahan (BBB) scale, which distinguishes between 22 progressive levels of recovery. As observed previously, recovery during the 6 weeks following injury was inversely related to the severity of injury. However, clenbuterol caused substantial enhancement of recovery of locomotor function at the two most severe levels of injury (BBB scores 10-12 vs 2-4). In addition, the extent of recovery was directly related to sparing of spinal cord tissue at the contusion center in both untreated and clenbuterol-treated spinal cords. Optimization of beta(2)-agonist treatment may lead to a useful therapeutic modality for treatment of spinal cord contusion injury.

Clenbuterol retards loss of motor function in motor neuron degeneration mice

Motor neuron degeneration (mnd) mice exhibit lysosomal accumulation of lipofuscin-like material that is associated with progressive loss of motor function and strength. Motor dysfunction scores at 8.5-9 months of age were highly correlated with the occurrence of abnormal spinal motor neurons with eccentric nuclei, although the total numbers of motor neurons were not significantly reduced. Nuclear eccentricity is a characteristic of the axon reaction that results from injury and subsequent compensatory axonal sprouting indicating axonal/synaptic dysfunction in mnd motor neurons. Treatment with clenbuterol, a beta(2)-adrenoceptor agonist that can enhance regeneration of motor neuron axons, opposed the development of motor deficits in parallel with a reduced proportion of motor neurons with eccentric nuclei consistent with improved synaptic function. Clenbuterol also opposed decreases in grip strength and muscle mass suggesting beta(2)-agonist treatment as a potential therapeutic modality for lipofuscinoses.

X-irradiation of the contusion site improves locomotor and histological outcomes in spinal cord-injured rats

We have determined whether X-irradiation of the injury site can oppose tissue loss and improve recovery of locomotor function following contusion injury of the spinal cord. Contusion injury was produced in rats at the level of T10 with a weight drop device. Localized X-irradiation (20 Gy) of the injury site was performed at 20 min and 1, 2, 4, 7, and 17 days postinjury. Locomotor recovery was then determined with the 21-point Basso, Beattie, and Bresnahan (BBB) scale. X-irradiation enhanced recovery of locomotor function during a subsequent 6-week observation period when administered 20 min and 1 or 2 days following contusion injury (final BBB score approximately 7-8). X-irradiation at 4-17 days postinjury did not significantly affect final locomotor scores compared with unirradiated rats (final BBB score approximately 2), in marked contrast to previous studies where X-irradiation applied only at 17-18 days benefitted transection injury. The extent of recovery was directly related to measurements of sparing of spinal cord tissue at the contusion center. Because the treatment time window occurred earlier in contusion than reported for transection injury, the results suggest that contusion injury rapidly initiates underlying radiation-sensitive processes that occur only following a delay of several weeks after transection injury. Further optimization of X-ray treatment may lead to a useful therapeutic modality for use in spinal cord contusion injury.

Proteasome- and p53-dependent Masking of Signal Transducer and Activator of Transcription (STAT) Factors

Hepatoma Hep3B cell lines stably expressing a temperature-sensitive p53 species (p53-Val-135) displayed a reduced response to interleukin-6 (IL-6) when cultured at the wild-type (wt) p53 temperature (Wang, L., Rayanade, R., Garcia, D., Patel, K., Pan, H., and Sehgal, P. B. (1995) J. Biol. Chem. 270, 23159-23165). We now report that in such cultures IL-6 caused a rapid (20-30 min) and marked loss of cellular immunostaining for STAT3 and STAT5, but not for STAT1. The loss of STAT3 and STAT5 immunostaining was transient (lasted 120 min) and tyrosine kinase-dependent, and even though the loss was blocked by the proteasome inhibitors MG132 and lactacystin it was not accompanied by changes in cellular levels of STAT3 and STAT5 proteins suggesting that IL-6 triggered a rapid masking but not degradation of these transcription factors. STAT3 and STAT5 masking was accompanied by a reduction in IL-6-induced nuclear DNA-binding activity. The data suggest that p53 may influence Jak-STAT signaling through a novel indirect mechanism involving a wt p53-dependent gene product which upon cytokine addition is activated into a “STAT-masking factor” in a proteasome-dependent step.

Improved functional recovery with oxandrolone after spinal cord injury in rats.

At present, only the corticosteroid, methylprednisolone, is used for acute spinal cord injury to improve function. However, improvements are modest, and are associated with myopathy and immunosuppression so that alternative treatments are needed. Oxandrolone is an androgenic steroid with potential neuroprotective properties that is used to prevent muscle loss and is not immunosuppressive. Oxandrolone increased locomotor recovery concomitant with reduced loss of cord tissue in a standard weight drop model of spinal cord contusion injury indicating oxandrolone as a possible alternative to methylprednisolone. Oxandrolone also increased axonal sprouting within the ventral horns distal to the injury consistent with formation of relay circuits mediating locomotor recovery.

STEREOTACTIC RADIOSURGERY IMPROVES LOCOMOTOR RECOVERY AFTER SPINAL CORD INJURY IN RATS

OBJECTIVECurrently, because of the precision of stereotactic radiosurgery, radiation can now be delivered by techniques that shape the radiation beam to the tissue target for a variety of clinical applications. This avoids unnecessary and potentially damaging irradiation of surrounding tissues inherent in conventional irradiation, so that irradiation of the minimum volume of tissue necessary for optimal therapeutic benefit can be achieved. Although conventional x-irradiation has been shown to improve recovery from spinal cord injury in animals, the efficacy of targeted irradiation of the injured spinal cord has not been demonstrated previously. The purpose of these studies was to determine whether stereotactic x-irradiation of the injured spinal cord can enhance locomotor function and spare spinal cord tissue after contusion injury in a standard experimental model of spinal cord injury. METHODSContusion injury was produced in rats at the level of T10 with a weight-drop device, and doses of x-irradiation were delivered 2 hours after injury via a Novalis, 6-MeV linear accelerator shaped beam radiosurgery system (BrainLAB USA, Westchester, IL) in 4 sequential fractions, with beam angles 60 to 70 degrees apart, at a rate of 6.4 Gy/minute. The target volume was a 4 × 15-mm cylinder along the axis of the spinal cord, with the isocenter positioned at the contusion epicenter. Locomotor function was determined for 6 weeks after injury with the 21-point Basso, Beattie, and Bresnahan (BBB) locomotor scale and tissue sparing in histological sections of the spinal cord. RESULTSLocomotor function recovered progressively during the 6-week postinjury observation period. BBB scores were significantly greater in the 10-Gy x-irradiated group compared with controls (9.4 versus 7.3; P < 0.05), indicating hind limb weight support or dorsal stepping in contrast to hind limb joint mobility without weight bearing. Doses in the range of 2 to 10 Gy increased BBB scores progressively, whereas greater doses of 15 to 25 Gy were associated with lower BBB scores. The extent of locomotor recovery after treatment with x-irradiation correlated with measurements of spared spinal cord tissue at the contusion epicenter. CONCLUSIONThese results suggest a beneficial role for stereotactic radiosurgery in a rat model of acute spinal cord contusion injury and raise hopes for human treatment strategies. Additional animal studies are needed to further define potential benefits.

Perfusion imaging of spinal cord contusion: injury-induced blockade and partial reversal by β2-agonist treatment in rats

OBJECT Traumatic injury to the spinal cord results in considerable delayed tissue loss. The authors investigated the extent to which ischemia occurs following contusion-induced spinal cord injury and whether ischemia exacerbates tissue damage that leads to the loss of locomotor function. They also determined if ischemia is reversed with β2-adrenoceptor agonist treatment, which has been established to be neuroprotective following contusion injury. METHODS The extent and role of circulation loss in spinal cord injury was determined in an established experimental model of contusion injury. The spinal cord dura mater of Wistar rats was exposed by performing a laminectomy at T-8 to T-11. Laser Doppler perfusion imaging was then used to measure microcirculation in the exposed spinal cord. After imaging, a moderately severe contusion injury was produced using a weight-drop device unto the exposed dura at T-10. Perfusion imaging was again performed, scans were quantitated, and integrated intensities were compared. RESULTS Postinjury imaging revealed an 18%-27% reduction in perfusion in regions rostral and caudal to the injury site, and a 68% reduction was observed at the contusion epicenter. These perfusion losses persisted for at least 48 hours. At 24 hours after injury, some rats were intraperitoneally injected with 2 mg/kg of the β2-adrenoceptor agonist clenbuterol, which has been shown to promote the partial recovery of locomotor function and spare spinal cord tissue when administered within 2 days after contusion injury. Clenbuterol injection caused a gradual increase in perfusion, which was detectable at 30 minutes postinjection and continued over time, resulting in an 127% overall increase in perfusion at the epicenter 24 hours after treatment. CONCLUSIONS These results suggest that the occurrence of chronic perfusion loss after contusion contributes to delayed damage and tissue loss. In contrast, β2-adrenoceptor agonist treatment may exert neuroprotection by restoring perfusion, thereby preventing ischemic neurodegeneration. The ability of laser Doppler imaging to measure the loss of perfusion and its restoration upon treatment suggests that it may have clinical utility in the assessment and treatment of spinal cord injury.