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• W2007185910 abstract "Despite the widespread use of polypeptide growth factors as pharmacological agents, little is known about the extent to which these molecules regulate their cognate cell surface receptors and signal transduction pathways in vivo. We have addressed this issue with respect to the neurotrophic molecule ciliary neurotrophic factor (CNTF). Administration of CNTF in vivo resulted in modest decreases in levels of CNTFRα mRNA and protein in skeletal muscle. CNTF causes the rapid tyrosine phosphorylation of LIFRβ and gp130 and the induction of the immediate-early gene, tis11; injection of CNTF 3-7 h after an initial exposure failed to re-stimulate these immediate-early responses, suggesting a biochemical desensitization to CNTF not accounted for by decreased receptor protein. To determine whether the desensitization of immediate-early responses caused by CNTF resulted in a functional desensitization, we compared the efficacy of multiple daily injections versus a single daily dose of CNTF in preventing the denervation-induced atrophy of skeletal muscle. Surprisingly, injections of CNTF every 6 h, which falls within the putative refractory period for biochemical responses, resulted in efficacy equal to or greater than injections once daily. These results suggest that although much of the CNTF signal transduction machinery is down-regulated with frequent CNTF dosing, biological signals continue to be recognized and interpreted by the cell. Despite the widespread use of polypeptide growth factors as pharmacological agents, little is known about the extent to which these molecules regulate their cognate cell surface receptors and signal transduction pathways in vivo. We have addressed this issue with respect to the neurotrophic molecule ciliary neurotrophic factor (CNTF). Administration of CNTF in vivo resulted in modest decreases in levels of CNTFRα mRNA and protein in skeletal muscle. CNTF causes the rapid tyrosine phosphorylation of LIFRβ and gp130 and the induction of the immediate-early gene, tis11; injection of CNTF 3-7 h after an initial exposure failed to re-stimulate these immediate-early responses, suggesting a biochemical desensitization to CNTF not accounted for by decreased receptor protein. To determine whether the desensitization of immediate-early responses caused by CNTF resulted in a functional desensitization, we compared the efficacy of multiple daily injections versus a single daily dose of CNTF in preventing the denervation-induced atrophy of skeletal muscle. Surprisingly, injections of CNTF every 6 h, which falls within the putative refractory period for biochemical responses, resulted in efficacy equal to or greater than injections once daily. These results suggest that although much of the CNTF signal transduction machinery is down-regulated with frequent CNTF dosing, biological signals continue to be recognized and interpreted by the cell. Ciliary neurotrophic factor (CNTF) 1The abbreviations used are: CNTFciliary neurotrophic factorCNTFRαciliary neurotrophic factor receptor αDRGdorsal root ganglionEDLextensor digitorum longusLIFleukemia inhibitory factorLIFRβleukemia inhibitory factor receptorMAP kinasemitogen-activated protein kinaseANOVAanalysis of variance. was originally described for its survival-promoting actions on parasympathetic (ciliary) neurons but has since been shown to exert trophic effects on a number of peripheral and central neurons, including motor, sensory, sympathetic, basal forebrain, hippocampal, and cerebellar neurons in vitro (1Ip N.Y. Yancopoulos G.D. Prog. Growth Factor Res. 1992; 4: 139-155Google Scholar). Studies on the physiological properties of CNTF in these culture systems have provided insights into the pharmacological potential of this factor in vivo. For example, motor neurons express CNTF receptors (2Ip N. McClain J. Barrezueta N.X. Aldrich T.H. Pan L. Li Y. Wiegand S.J. Friedman B. Davis S. Yancopoulos G.D. Neuron. 1993; 10: 89-102Google Scholar) and retrogradely accumulate 125I-CNTF (3Curtis R. Adryan K.M. Zhu Y. Harkness P.J. Lindsay R.M. DiStefano P.S. Nature. 1993; 365: 253-255Google Scholar) in a receptor-mediated fashion. Accordingly, CNTF prevents motor neuron cell death and ameliorates neuromuscular deficits in wobbler and pmn mutant mice, both models of motor neuron disease (4Mitsumoto H. Ikeda K. Klinkosz B. Cedarbaum J.M. Wong V. Lindsay R.M. Science. 1994; 265: 1107-1110Google Scholar, 5Sendtner M. Schmalbruch H. Stückli K.A. Carroll P. Kreutzberg G.W. Thoenen H. Nature. 1992; 358: 502-504Google Scholar). In the central nervous system, CNTF prevents the axotomy-induced retrograde neuronal death of anterior thalamic neurons (6Clatterbuck R.E. Price D.L. Koliatsos V.E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2222-2226Google Scholar) and of neurons in the substantia nigra (7Hagg T. Quon D. Higaki J. Varon S. Neuron. 1992; 8: 145-158Google Scholar). Although CNTF receptors are highly localized in nervous tissues (2Ip N. McClain J. Barrezueta N.X. Aldrich T.H. Pan L. Li Y. Wiegand S.J. Friedman B. Davis S. Yancopoulos G.D. Neuron. 1993; 10: 89-102Google Scholar), receptor expression has also been characterized in many nonneuronal tissues (8MacLennan A.J. Gaskin A.A. Lado D.C. Mol. Brain Res. 1994; 25: 251-256Google Scholar, 9DiStefano P.S. Stark J. Adryan K.M. Curtis R. Farruggella T. Stahl N. Lindsay R.M. Soc. Neurosci. Abstr. 1994; 20: 42Google Scholar), suggesting a wider spectrum of action of this factor in vivo than was initially thought. Most prominently, functional CNTF receptors have been localized to skeletal muscle, and CNTF displays trophic activity on denervated muscle (10Helgren M.E. Squinto S.P. Davis H.L. Parry D.J. Boulton T.G. Heck C.S. Zhu Y. Yancopoulos G.D. Lindsay R.M. DiStefano P.S. Cell. 1994; 76: 493-504Google Scholar). In this model, administration of CNTF partially prevents the loss of muscle wet weight, the decreased myofiber cross-sectional area, and the deficits in tensile properties as a result of muscle denervation. ciliary neurotrophic factor ciliary neurotrophic factor receptor α dorsal root ganglion extensor digitorum longus leukemia inhibitory factor leukemia inhibitory factor receptor mitogen-activated protein kinase analysis of variance. The functional receptor for CNTF exists as a tripartite complex consisting of CNTFRα, a glycosylphosphatidylinositol-anchored protein, and two β components termed leukemia inhibitory factor receptor-β (LIFRβ) and gp130 (11Davis S. Aldrich T.H. Stahl N. Pan L. Taga T. Kishimoto T. Ip N.Y. Yancopoulos G.D. Science. 1993; 260: 1805-1808Google Scholar, 12Stahl N. Yancopoulos G.D. Cell. 1993; 74: 587-590Google Scholar). These β components are also necessary receptor subunits for LIF, oncostatin M, interleukin-6, and cardiotrophin-1 (13Stahl N. Yancopoulos G.D. J. Neurobiol. 1994; 25: 1454-1466Google Scholar, 14Pennica D. Shaw K.J. Swanson T.A. Moore M.W. Shelton D.L. Zioncheck K.A. Rosenthal A. Taga T. Paoni N.F. Wood W.I. J. Biol. Chem. 1995; 270: 10915-10922Google Scholar), molecules whose tertiary structures are related to that of CNTF (12Stahl N. Yancopoulos G.D. Cell. 1993; 74: 587-590Google Scholar, 15Bazan J.F. Neuron. 1991; 7: 197-208Google Scholar, 16Robinson R.C. Grey L.M. Staunton D. Vankelecom H. Vernallis A.B. Moreau J.-F. Stuart D.I. Heath J.K. Jones E.Y. Cell. 1994; 77: 1101-1116Google Scholar, 17McDonald N.Q. Panayotatos N. Hendrickson W.A. EMBO J. 1995; 14: 2689-2699Google Scholar). The binding of CNTF to CNTFRα results in the stepwise recruitment of gp130 and LIFRβ on the cell surface, forming a high affinity complex capable of initiating signal transduction cascades (13Stahl N. Yancopoulos G.D. J. Neurobiol. 1994; 25: 1454-1466Google Scholar). Unlike the receptor tyrosine kinase class of receptors, formation of the CNTF receptor complex results in tyrosine phosphorylation of the β subunits by the Jak/Tyk family of cytoplasmic tyrosine kinases that are constitutively associated with the β subunits (18Stahl N. Boulton T.G. Farruggella T. Ip N.Y. Davis S. Witthuhn B.A. Quelle F.W. Silvennoinen O. Barbieri G. Pellegrini S. Ihle J.N. Yancopoulos G.D. Science. 1994; 263: 92-95Google Scholar). In addition, autophosphorylation of Jak occurs on distinct tyrosine residues. Specific tyrosine phosphate residues on the β subunits and the Jaks serve as docking sites for certain SH2 domain-containing proteins, the combinations of which may dictate responsiveness to various growth and trophic factors (18Stahl N. Boulton T.G. Farruggella T. Ip N.Y. Davis S. Witthuhn B.A. Quelle F.W. Silvennoinen O. Barbieri G. Pellegrini S. Ihle J.N. Yancopoulos G.D. Science. 1994; 263: 92-95Google Scholar). Immediate-early signal transduction pathways in response to CNTF proceed via activation of the MAP kinase pathway, as well as by activation of the STAT family of cytosolic transcriptional activators (see Ref. 19Curtis R. DiStefano P.S. Trends Cell Biol. 1994; 4: 383-386Google Scholar). This, in turn, results in the induction of immediate-early genes such as tis11 and c-fos, which have been shown to contain in their 5′-untranslated regions a CNTF response element, which most likely represents a STAT response element (20Bonni A. Frank D.A. Schindler C. Greenberg M.E. Science. 1993; 262: 1575-1579Google Scholar). These signal transduction cascades have been well established in various CNTF-responsive cells in vitro (21Ip N.Y. Nye S.H. Boulton T.G. Davis S. Taga T. Li Y. Birren S.J. Yasukawa K. Kishimoto T. Anderson D.J. Stahl N. Yancopoulos G.D. Cell. 1992; 69: 1121-1132Google Scholar), and we have shown recently that CNTF administration in vivo results in the phosphorylation of gp130/LIFRβ and induction of tis11 in a number of CNTFRα-bearing tissues, including skeletal muscle, dorsal root ganglia, peripheral nerve, adrenal, and heart tissues (9DiStefano P.S. Stark J. Adryan K.M. Curtis R. Farruggella T. Stahl N. Lindsay R.M. Soc. Neurosci. Abstr. 1994; 20: 42Google Scholar, 10Helgren M.E. Squinto S.P. Davis H.L. Parry D.J. Boulton T.G. Heck C.S. Zhu Y. Yancopoulos G.D. Lindsay R.M. DiStefano P.S. Cell. 1994; 76: 493-504Google Scholar). The potent survival-promoting and neuroprotective actions of trophic factors have stimulated considerable interest in their potential as therapeutic agents in neurological disorders such as trauma, neurodegeneration, and muscle disease (22Lindsay R.M. Wiegand S.J. Altar C.A. DiStefano P.S. Trends Neurosci. 1994; 17: 182-190Google Scholar). To achieve maximal benefit from such molecules, it becomes important to determine their optimal dose as well as their optimal dose frequency. Although ligand-induced down-regulation of receptors is a common feature of neurotransmitters, neuropeptides, and certain polypeptide growth factors, less is known about the extent to which repeated or chronic administration of polypeptide growth factors may induce desensitization of their signal transduction pathways and/or the downstream functional sequelae. In the present study we have addressed several critical questions regarding the administration of pharmacological doses of CNTF in vivo. First, does CNTF cause decreased expression of CNTFRα mRNA or protein in responsive tissues? Second, are immediate-early cellular responses (tyrosine phosphorylation or tis11 induction) also desensitized or down-regulated with repeated administration of CNTF? Third, is there cross-desensitization between CNTF and related cytokines, such as LIF? Fourth and most importantly, is down-regulation of receptor or of signal transduction cascades associated with a functional desensitization to repeated administration of the factor? We show here that CNTF administration results in decreased levels of CNTF receptor, and repeated frequent administration results in reduced tyrosine phosphorylation of receptor components and induction of tis11. To assess whether these events are associated with a functional desensitization to CNTF, we examined the ability of multiple, frequent doses of CNTF to prevent the denervation-induced atrophy of skeletal muscle, a property of CNTF established previously in our laboratory (10Helgren M.E. Squinto S.P. Davis H.L. Parry D.J. Boulton T.G. Heck C.S. Zhu Y. Yancopoulos G.D. Lindsay R.M. DiStefano P.S. Cell. 1994; 76: 493-504Google Scholar). We show that despite down-regulation of the CNTF receptor and its signal transduction cascades, repeated frequent administration of CNTF resulted in no functional desensitization to this factor, establishing a non-equivalence between biochemical desensitization and pharmacological activity of CNTF in vivo. These studies suggest a model whereby tissues maintain their responsiveness to a given factor by adjusting the intracellular “gain” to increased or persistent extracellular signals. Male Sprague-Dawley rats weighing 250-300 g were housed two per cage, given food and water ad libitum, and maintained on a 12-h light-dark cycle. Recombinant human CNTF (referred to as CNTF henceforth) was formulated in 10 mM phosphate, 12 mM lactate, 5% mannitol, pH 4.5. Recombinant human LIF (generously provided by Dr. J. A. Miller, Amgen) was supplied in phosphate-buffered saline. To examine in vivo signal transduction events, animals were injected with lactate/phosphate/mannitol vehicle, CNTF, or LIF at doses ranging from 0.1-1.0 mg/kg. In all experimental paradigms, CNTF, LIF, and vehicle solutions were administered subcutaneously. It was determined previously that tyrosine phosphorylation and tis11 induction peaked 45 and 60 min, respectively, after a subcutaneous dose of CNTF (10Helgren M.E. Squinto S.P. Davis H.L. Parry D.J. Boulton T.G. Heck C.S. Zhu Y. Yancopoulos G.D. Lindsay R.M. DiStefano P.S. Cell. 1994; 76: 493-504Google Scholar). Therefore, rats were sacrificed 52 min after the injection, and skeletal muscle (soleus and extensor digitorum longus (EDL)) and other tissues were rapidly dissected; routinely, total RNA was prepared from the right muscle, and the left muscle was used for gp130/LIFRβ tyrosine phosphorylation analysis. In some experiments, animals received sciatic nerve transections 24 h prior to CNTF administration to determine the effect of denervation on immediate-early responses to CNTF. To assess possible refractory periods for phosphorylation or tis11 induction after repeated doses of CNTF, the following experimental paradigm was adopted. Rats were administered a pre-dose of either vehicle or CNTF, followed 1-48 h later by a test dose of CNTF. The doses of CNTF used were either 0.1 or 1.0 mg/kg. Fifty-two min after the test dose, animals were sacrificed and the muscles or lumbar 1-6 dorsal root ganglia (L1-L6 DRG) were processed for immediate-early responses as described above (see also Fig. 3). For cross-desensitization experiments, LIF (0.1 mg/kg) was also included in the pre-dose and test dose paradigm. For controls, pre-dose injections of vehicle were made 5 h prior to the test dose. Soleus muscles or L1-L6 DRG were homogenized in 4 M guanidine containing 5%β-mercaptoethanol, and total RNA was prepared by the method of Chomszynski and Sacchi (23Chomszynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Google Scholar). Ten µg of total RNA was separated on 1% formaldehyde gels and transferred to nylon membranes. Membranes were hybridized with 32P-labeled cDNA probes for rat CNTFRα or tis11 as described (2Ip N. McClain J. Barrezueta N.X. Aldrich T.H. Pan L. Li Y. Wiegand S.J. Friedman B. Davis S. Yancopoulos G.D. Neuron. 1993; 10: 89-102Google Scholar, 10Helgren M.E. Squinto S.P. Davis H.L. Parry D.J. Boulton T.G. Heck C.S. Zhu Y. Yancopoulos G.D. Lindsay R.M. DiStefano P.S. Cell. 1994; 76: 493-504Google Scholar). Hybridizing species were identified using Kodak X-O-MAT film (Eastman Kodak Co., Rochester, NY). CNTF was radioiodinated to a specific activity of ~2000 cpm/fmol as described previously (3Curtis R. Adryan K.M. Zhu Y. Harkness P.J. Lindsay R.M. DiStefano P.S. Nature. 1993; 365: 253-255Google Scholar). Muscles from rats treated with CNTF were homogenized with a polytron in 10 volumes of phosphate-buffered saline containing 10 µg/ml aprotinin and 1 mM PMSF. Samples were centrifuged 1,000 × g for 10 min and the resulting supernatants were centrifuged 100,000 × g for 20 min in a Beckman ultracentrifuge (model TL100). The resulting pellets were resuspended in the homogenization buffer and 200 µl aliquots of membranes (containing equal protein concentrations) were incubated with 4 nM 125I-CNTF in the presence or absence of a 200-fold excess of unlabeled rat CNTF to determine specific binding. After binding for 1.5 h on ice, the cross-linking agent disuccinimidyl suberate (200 µM) was added to each tube for 30 min at room temperature. Cross-linking was quenched by adding 1 ml of 20 mM Tris, 0.15 M NaCl, pH 7.4, for 15 min on ice. Samples were clarified by centrifugation at 100,000 × g for 20 min. Pellets were resuspended in the Tris/NaCl quenching buffer and 370 milliunits of phosphatidylinositol-specific phospholipase C (Sigma) were added to each sample for 30 min at 37°C to cleave the glycosylphosphatidylinositol-anchored CNTFRα from membranes. Samples were clarified by ultracentrifugation and the supernatants were incubated overnight at 4°C with 25 µg of recombinant gp130-IgG produced in baculovirus. 2T. E. Ryan, unpublished data. 125I-CNTF-CNTFRα-gp130-IgG complexes were precipitated with protein G-Sepharose and pellets were washed three times with phosphate-buffered saline/0.2% Tween-20. Samples were resolved on 7.5% acrylamide SDS gels and 125I-CNTF-cross-linked species were visualized autoradiographically using Kodak BioMax MR film. Immunoprecipitations and antiphosphotyrosine immunoblots for gp130/LIFRβ/Jak complexes were performed essentially as described (10Helgren M.E. Squinto S.P. Davis H.L. Parry D.J. Boulton T.G. Heck C.S. Zhu Y. Yancopoulos G.D. Lindsay R.M. DiStefano P.S. Cell. 1994; 76: 493-504Google Scholar, 24Boulton T.G. Stahl N. Yancopoulos G.D. J. Biol. Chem. 1994; 269: 11648-11655Google Scholar, 25Stahl N. Davis S. Wong V. Taga T. Kishimoto T. Ip N.Y. Yancopoulos G.D. J. Biol. Chem. 1993; 268: 7628-7631Google Scholar). Autoradiographic bands from Northern blots, Western blots, or cross-linking experiments were quantified using an LKB (Uppsala, Sweden) laser densitometer (model XL) interfaced with the GelScan XL program (LKB) to integrate area under the curve for individual bands. In instances in which band intensities were beyond the linear range of detection, band intensities from multiple exposures were analyzed to determine relative band intensities. In vivo responses to CNTF were assessed by monitoring the degree to which CNTF prevented the denervation-induced atrophy of soleus muscle. Briefly, animals were anesthetized with a mixture of ketamine/xylazine (50/10 mg/kg, intraperitoneal) and a 5-mm segment of the right sciatic nerve was resected at the level of the knee. The left side was sham operated. Animals were administered vehicle or CNTF (0.1-0.3 mg/kg) either once daily or four times daily every 6 h for 4 days. The animals were sacrificed, the muscles were weighed, and the ratios of right:left muscle weights were determined for each animal. The percent rescue of muscle weight was also calculated based on wet weight ratios, using the following formula: Treatmentratio- Mean Vehicleratio/1 - Mean Vehicleratio. Differences between treatment groups were compared using analysis of variance (ANOVA) and Dunnett's post hoc test. All animal use in this study was conducted in compliance with approved institutional animal care and use protocols and according to NIH guidelines (52NIH, (1985) Guide for the Care and Use of Laboratory Animals, NIH Publication No. 86-23.Google Scholar). A series of experiments was carried out to determine whether administration of CNTF caused down-regulation of CNTFRα mRNA and protein in vivo. Given that CNTFRα mRNA is dramatically up-regulated in skeletal muscle within 24 h after denervation, we examined the ability of CNTF to regulate its own receptor expression both in normal and in denervated muscles. Rats were injected once daily with 1.0 mg/kg CNTF for 1, 4, or 7 days, and the steady state levels of CNTFRα mRNA were determined in soleus and EDL muscles. A single injection of CNTF resulted in significant down-regulation of CNTFRα mRNA expression in both soleus and EDL muscles 24 h later (Fig. 1, A and B). When rats were injected daily for 4 or 7 days, the down-regulation of receptor was maintained. This was apparent in normal, as well as in denervated, muscles (Fig. 1, A and B). Fig. 1, C and D, shows the quantification of CNTFRα mRNA down-regulation in response to daily CNTF administration. The results are from multiple experiments and include data for soleus and EDL muscles. With the exception of 1 day after sciatic nerve lesion, steady state expression of CNTFRα was significantly reduced to approximately 50% of control throughout the treatment period (p < 0.05, ANOVA). Further investigation revealed that a single injection of CNTF resulted in decreased CNTFRα mRNA as early as 1 h after injection (not shown). In addition, two injections placed 5 h apart resulted in a significant reduction (65%) in mRNA (Fig. 1E). In situ hybridization studies in muscle confirmed the down-regulation of CNTFRα mRNA by CNTF treatment (data not shown). We next used a 125I-CNTF cross-linking method to estimate relative levels of CNTFRα protein on skeletal muscle membranes after daily exposure to CNTF. Fig. 2A shows that this cross-linking assay was specific for CNTFRα because 1) the band comigrated with the known molecular mass (~100 kDa) for the CNTF-CNTFRα complex (25Stahl N. Davis S. Wong V. Taga T. Kishimoto T. Ip N.Y. Yancopoulos G.D. J. Biol. Chem. 1993; 268: 7628-7631Google Scholar); 2) cross-linking was blocked by a 200-fold excess of unlabeled CNTF; 3) omitting the phosphatidylinositol-specific phospholipase C step or the gp130-IgG incubation resulted in no signal; and 4) a recombinant CNTFRα (with a slightly higher molecular mass) competed for cross-linking of 125I-CNTF to muscle membrane CNTFRα (not shown). As with CNTFRα mRNA, CNTF induced a down-regulation of CNTFRα protein reproducibly, although the time course was much more protracted and the magnitude of the response was not as great (Fig. 2, B and C). Denervation resulted in a steady increase in 125I-CNTF cross-linked to CNTFRα, reaching 4-fold by 7 days (Fig. 2, A-C). Significant down-regulation (35-40% reduction) was observed only after 7 days of treatment in normal muscle, and receptor was decreased by 40 and 15% after 1 or 7 days, respectively, in denervated soleus muscle. Similar quantitative results were observed with EDL and gastrocnemius muscles. We also tested whether three injections of CNTF, placed 5 h apart, would result in decreased CNTFRα, as determined by 125I-CNTF cross-linking to muscle membranes. This treatment resulted in modest decreases of only 20% in normal muscles (not shown). This contrasts with the substantial down-regulation of CNTFRα mRNA with short-interval, repeated dosing (Fig. 1E). We have previously shown that subcutaneous administration of CNTF results in increased tyrosine phosphorylation of LIFRβ, gp130, and Jak kinase in skeletal muscle (10Helgren M.E. Squinto S.P. Davis H.L. Parry D.J. Boulton T.G. Heck C.S. Zhu Y. Yancopoulos G.D. Lindsay R.M. DiStefano P.S. Cell. 1994; 76: 493-504Google Scholar). This effect peaks at 45 min and returns to baseline 2-3 h postinjection. Shortly after the onset of tyrosine phosphorylation, the immediate-early gene tis11 is induced, peaking at 1 h and extinguishing by ~4 h. The half-life of CNTF is short, and doses of 0.1-1.0 mg/kg are cleared from the plasma by 2-4 h. Experiments were undertaken to determine whether repeated doses of CNTF, administered at different time intervals, resulted in down-regulation (desensitization) of these immediate-early responses in skeletal muscle. Rats were administered a pre-dose of either vehicle or CNTF (1.0 mg/kg), followed 1, 3, 5, 7, 24, or 48 h later by a test dose of CNTF (see “Materials and Methods” and Fig. 3). Consistent with previous studies, administration of CNTF resulted in increased tyrosine phosphorylation of LIFRβ and gp130 52 min after the injection (Fig. 4). Performing this experiment in rats whose muscles were denervated by sciatic nerve transection resulted in generally heightened responses. If the animals were injected with CNTF prior to the test dose (and sacrificed at 52 min), there was a marked diminution of the response to the test dose. This was slightly evident at the 1-h interval, and with 3-, 5-, and 7-h intervals the response was greatly diminished. It is important to note that tyrosine phosphorylation of receptor components in this paradigm never reached the background levels observed in vehicle-treated rats. When the dosing interval was spaced 24 or 48 h apart there was no desensitization, indicating that the apparent refractory period for this response was between 7 and 24 h. We next determined whether a similar pattern of desensitization was observed for tis11 induction using the same pre-dose/test dose paradigm outlined in Fig. 3. Using a dose of 1.0 mg/kg CNTF, tis11 was induced 20-fold in normal soleus muscle, and prior denervation resulted in a 40-fold induction (Fig. 5). As with tyrosine phosphorylation experiments, administration of CNTF at 1, 3, 5, or 7 h prior to the test dose caused a progressive failure of CNTF to elicit increased tis11 responses. Furthermore, the response returned to its maximum when the interval between doses was 24-48 h. Quantification of this response in normal and denervated muscles is represented in Fig. 6. It is once again important to note that even though the desensitization of the tis11 response is quite substantial, it was never reduced to levels observed in vehicle-treated animals and was routinely 2.5 ± 0.4-fold above background (p < 0.05; n = 3). Similar quantitative results were obtained from densitometric analysis of tyrosine phosphorylation experiments (not shown). We then estimated the dose of CNTF necessary to elicit these small increments (2.5-fold) in receptor tyrosine phosphorylation or tis11 induction to correlate this with the levels of CNTF that might be encountered by tissues under physiological conditions. Therefore, we performed a dose-response test with CNTF in normal and sciatic nerve-transected rats. Fig. 7 shows that a dose of 0.01 mg/kg caused a discernible induction of tis11 in normal rats and that doses of 0.001-0.01 mg/kg were effective in previously denervated muscle; the maximal activation of tis11 was also greater in muscles that had been denervated 24 h prior to injection (Fig. 6, Fig. 7). From the results presented thus far there are at least two possible mechanisms by which desensitization might arise. First, the fact that administration of CNTF results in down-regulation of receptor mRNA and protein suggests that the desensitization mechanism lies at the level of the cell surface receptor. Second, it is possible that other downstream regulatory events are involved. To address the latter possibility, we performed cross-desensitization experiments with LIF and CNTF, using the pre-dose/test dose paradigm (Fig. 3) with tis11 induction as the readout. We took advantage of the fact that LIF also uses components of the CNTF receptor complex, namely LIFRβ and gp130, but does not bind to CNTFRα. In addition, LIF and other related factors activate signal transduction cascades similar to those elicited by CNTF (21Ip N.Y. Nye S.H. Boulton T.G. Davis S. Taga T. Li Y. Birren S.J. Yasukawa K. Kishimoto T. Anderson D.J. Stahl N. Yancopoulos G.D. Cell. 1992; 69: 1121-1132Google Scholar). Using DRG as the responsive tissue, it was found that a pre-dose of LIF (0.1 mg/kg, 4 h earlier) resulted in biochemical desensitization to a test dose of CNTF (0.1 mg/kg). However, the inverse was not true in that tissues preexposed to CNTF were not refractory to a test dose of LIF (Fig. 8A). Similar results were observed in soleus muscle (Fig. 8B). Therefore, mechanisms downstream from the receptor may be involved in the biochemical desensitization with repeated administration of CNTF. Given that CNTF treatment caused down-regulation of CNTFRα and marked desensitization of immediate-early signal transduction responses, we sought to determine whether this resulted in a functional desensitization to CNTF. To assess this, we tested various dosing regimens on the ability of CNTF to prevent the denervation-induced atrophy of skeletal muscle. We have previously shown that treatment of rats daily with doses of CNTF (0.03-1.0 mg/kg) partially prevents this atrophy (10Helgren M.E. Squinto S.P. Davis H.L. Parry D.J. Boulton T.G. Heck C.S. Zhu Y. Yancopoulos G.D. Lindsay R.M. DiStefano P.S. Cell. 1994; 76: 493-504Google Scholar). To best approximate conditions that would down-regulate CNTF receptor and induce desensitization to immediate-early responses, rats were treated with vehicle or CNTF (0.1 or 0.3 mg/kg) every 6 h for 4 days; for comparison, other groups were treated once daily for 4 days. Rats whose muscles were denervated for 4 days showed a 30% reduction of wet weight, and once-daily dosing with 0.3 mg/kg CNTF resulted in only a 15-20% reduction in weight, or a rescue of 30% (Fig. 9, A and B). Surprisingly, treatment of rats with CNTF every 6 h resulted in equal or slightly greater efficacy on sparing of muscle wet weight (and a wet weight rescue of 50%) compared to those animals treated once daily (Fig. 9, A and B). It is worth noting that the muscle wet weight sparing effect was not the result of a single exposure to CNTF because discontinuation of the CNTF treatment resulted in a loss of the efficacy (0.70 ± 0.03 versus 0.73 ± 0.05; p = 0.6). Therefore, signals elicited by frequent injections of CNTF are critical to the efficacy, even though certain signal trans" @default.
• W2007185910 created "2016-06-24" @default.
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• W2007185910 title "Ciliary Neurotrophic Factor Induces Down-regulation of Its Receptor and Desensitization of Signal Transduction Pathways" @default.
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