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Muscle and bone are intimately linked by bi-directional signals regulating both muscle and bone cell gene expression and proliferation. It is generally accepted that muscle cells secrete factors (myokines) that influence adjacent bone cells, but these myokines are yet to be identified. We have previously shown that osteocyte-specific deletion of the co-receptor subunit utilized by IL-6 family cytokines, glycoprotein 130 (gp130), resulted in impaired bone formation in the trabecular bone, but enhanced periosteal expansion, suggesting a gp130-dependent periosteum-specific inhibition of osteoblast function, potentially induced by the local muscle fibres. We report here that differentiated primary calvarial osteoblasts cultured in myotube-conditioned media (CM) from myogenic C2C12 cells show reduced mRNA levels of genes associated with osteoblast differentiation. Alkaline phosphatase protein activity and all mRNA markers of osteoblast differentiation in the tested panel (runx2, osterix, alkaline phosphatase, parathyroid hormone (PTH) receptor, osteoprotegerin, osteocalcin, sclerostin) were reduced following culture with myotube CM. The exception was RANKL, which was significantly elevated in differentiated primary osteoblast cultures expressing osteocytic genes. A cytokine array of the C2C12 myotube-conditioned media identified TIMP-1 and MCP-1 as the most abundant myokines, but treatment with recombinant TIMP-1 or MCP-1 did not inhibit osteoblast gene expression. Rather, the IL-6 family cytokine ciliary neurotrophic factor (CNTF), which we found abundantly expressed by mouse muscle at the transcript and protein level, reduced osteoblast gene expression, although not to the same extent as the myotube-conditioned media. These data indicate that muscle cells secrete abundant TIMP-1, MCP-1, and CNTF, and that of these, only CNTF has the ability to suppress osteoblast function and gene expression in a similar manner to myotube-conditioned medium. This suggests that CNTF is an inhibitory myokine for osteoblasts.
Copyright © 2014 Elsevier Inc. All rights reserved.
Reprogramming of pancreatic exocrine cells into cells resembling beta cells may provide a strategy for treating diabetes. Here we show that transient administration of epidermal growth factor and ciliary neurotrophic factor to adult mice with chronic hyperglycemia efficiently stimulates the conversion of terminally differentiated acinar cells to beta-like cells. Newly generated beta-like cells are epigenetically reprogrammed, functional and glucose responsive, and they reinstate normal glycemic control for up to 248 d. The regenerative process depends on Stat3 signaling and requires a threshold number of Neurogenin 3 (Ngn3)-expressing acinar cells. In contrast to previous work demonstrating in vivo conversion of acinar cells to beta-like cells by viral delivery of exogenous transcription factors, our approach achieves acinar-to-beta-cell reprogramming through transient cytokine exposure rather than genetic modification.
Neuronal and axonal degeneration results in irreversible neurological disability in multiple sclerosis (MS) patients. A number of adaptive or neuroprotective mechanisms are thought to repress neurodegeneration and neurological disability in MS patients. To investigate possible neuroprotective pathways in the cerebral cortex of MS patients, we compared gene transcripts in cortices of six control and six MS patients. Out of 67 transcripts increased in MS cortex nine were related to the signalling mediated by the neurotrophin ciliary neurotrophic factor (CNTF). Therefore, we quantified and localized transcriptional (RT-PCR, in situ hybridization) and translational (western, immunohistochemistry) products of CNTF-related genes. CNTF-receptor complex members, CNTFRalpha, LIFRbeta and GP130, were increased in MS cortical neurons. CNTF was increased and also expressed by neurons. Phosphorylated STAT3 and the anti-apoptotic molecule, Bcl2, known down stream products of CNTF signalling were also increased in MS cortical neurons. We hypothesize that in response to the chronic insults or stress of the pathogenesis of multiple sclerosis, cortical neurons up regulate a CNTF-mediated neuroprotective signalling pathway. Induction of CNTF signalling and the anti-apoptotic molecule, Bcl2, thus represents a compensatory response to disease pathogenesis and a potential therapeutic target in MS patients.
PURPOSE - Application of ciliary neurotrophic factor (CNTF) can rescue mature photoreceptors from lesion-induced and hereditary degeneration. In the chick retina, expression of the CNTF receptor is present in a subpopulation of photoreceptor cells. The present study was undertaken to identify the CNTF receptor-expressing photoreceptors and to describe the subcellular localization of the receptor protein.
METHODS - The localization of the CNTF receptor was analyzed by light and electron microscopic immunocytochemistry in chick retinal wholemount preparations, with an antibody for CNTF receptor alpha (CNTFRalpha). Immunoreactive cells were identified by double labeling with immunocytochemical markers for photoreceptor subpopulations.
RESULTS - The CNTFRalpha antibody labeled evenly distributed outer segments (OS) of a photoreceptor subpopulation. CNTFRalpha-positive OS were associated with oil droplets of uniform size. Receptor immunoreactivity did not colocalize with markers for rods and red-green cones. Complete overlap was found after double labeling with the antibody CERN 933, which recognizes violet-sensitive cones in the chick retina. Ultrastructurally, the CNTFRalpha-immunoreactive OS showed rodlike properties: an elongated shape and stacks of membrane discs separated from the plasma membrane. Immunoreactivity was completely restricted to the plasma membrane of the OS and the inner membrane sheet of the photoreceptor calices present in avian retinas.
CONCLUSIONS - CNTFRalpha expression identifies a unique type of photoreceptors in the avian retina which does not fit into the classic morphologic definition of rods and cones. The specific expression in violet-sensitive photoreceptors suggests that CNTF may have a neuroprotective role related to the specific function of these cells.
Ciliary neurotrophic factor (CNTF) promotes the survival and differentiation of various neuronal and glial cell populations in the nervous system of vertebrates. In mammals, the ligand-binding alpha-subunit of the CNTF receptor (CNTFRalpha) is expressed in a variety of neuronal populations, including all CNTF-responsive cells. Previous studies suggested that functional differences in the CNTF/CNTF receptor system between chicks and mammals exist. The purpose of the present study was to examine the temporal and spatial expression pattern of the chick CNTFRalpha protein during CNS development. Receptor expression was detectable by immunoblotting in all CNS areas tested but showed area-specific developmental regulation. Interestingly, two variants of CNTFRalpha, 69 and 65 kD, were identified by immunoblotting with a shift from the higher to the lower molecular mass species occurring during development. Immunoreactivity for CNTFRalpha protein was preferentially observed in neuropil and white matter structures of the developing CNS while neuronal somata generally appeared unlabeled. For example, expression was observed in the olfactory system, in the telencephalon, in parts of the somatosensory system, in components of the tectofugal pathway, in the cerebellum, and in auditory brainstem nuclei. Fiber tracts that exhibit CNTFRalpha immunoreactivity were the lateral forebrain bundle, occipitomesencephalic tract, quintofrontal tract, and vestibular nerve. Our study identifies potential new targets of a chick CNTF-related molecule and reveals significant regional differences of CNTFRalpha protein expression between chick and mammals. These results suggest that the CNTF receptor performs distinct developmental functions in different animals.
Copyright 2003 Wiley-Liss, Inc.
Previous studies suggest that ciliary neurotrophic factor (CNTF) may represent one of the extrinsic signals controlling the development of vertebrate retinal photoreceptors. In dissociated cultures from embryonic chick retina, exogenously applied CNTF has been shown to act on postmitotic rod precursor cells, resulting in an two- to fourfold increase in the number of cells acquiring an opsin-positive phenotype. We now demonstrate that the responsiveness of photoreceptor precursors to CNTF is confined to a brief phase between their final mitosis and their terminal differentiation owing to the temporally restricted expression of the CNTF receptor (CNTFR alpha). As shown immunocytochemically, CNTFR alpha expression in the presumptive photoreceptor layer of the chick retina starts at embryonic day 8 (E8) and is rapidly down-regulated a few days later prior to the differentiation of opsin-positive photoreceptors, both in vivo and in dissociated cultures from E8. We further show that the CNTF-dependent in vitro differentiation of rods is followed by a phase of photoreceptor-specific apoptotic cell death. The loss of differentiated rods during this apoptotic phase can be prevented by micromolar concentrations of retinol. Our results provide evidence that photoreceptor development depends on the sequential action of different extrinsic signals. The time course of CNTFR alpha expression and the in vitro effects suggest that CNTF or a related molecule is required during early stages of rod differentiation, while differentiated rods depend on additional protective factors for survival.
Ciliary neurotrophic factor (CNTF) exerts a multiplicity of effects on a broad spectrum of target cells, including retinal neurons. To investigate how this functional complexity relates to the regulation of CNTF receptor alpha (CNTFR alpha) expression, we have studied the developmental expression of the receptor protein in chick retina by using immunocytochemistry. During the course of development, the receptor is expressed in all retinal layers, but three levels of specificity can be observed. First, the expression is regulated temporally with immunoreactivity observed in ganglion cells (embryonic day 8 [E8] to adult), photoreceptor precursors (E8-E12), amacrine cells (E10 to adult), bipolar cells (E12-E18), differentiated rods (E18 to adult), and horizontal cells (adult). Second, expression is restricted to distinct subpopulations of principal retinal neurons: preferentially, large ganglion cells; subpopulations of amacrine cells, including a particular type of cholinergic neuron; a distinctly located type of bipolar cell; and rod photoreceptors. Third, expression exhibits subcellular restriction: it is confined largely to dendrites in mature amacrine cells and is restricted entirely to outer segments in mature rods. These data correlate with CNTF effects on the survival of ganglion cells and mature photoreceptors, the in vitro differentiation of photoreceptor precursors and cholinergic amacrine cells, and the number of bipolar cells in culture described here or in previous studies. Thus, our results demonstrate an exceptional degree of complexity with respect to the regulation of neuronal CNTFR alpha expression in a defined model system. This suggests that the same signaling pathway is used to mediate a variety of regulatory influences, depending on the developmental stage and cell type.
The development of photoreceptors in the mammalian retina is thought to be controlled by extrinsic signals. We have shown previously that ciliary neurotrophic factor (CNTF) potently inhibits photoreceptor differentiation in cultures of rat retina. The present study analyzes which developmental processes are affected by CNTF. Rod differentiation as determined by opsin and recoverin immunocytochemistry was effectively blocked by CNTF and leukemia inhibitory factor, but not by other neurotrophic agents tested. CNTF did not influence proliferation, cell death, or survival, and had no effect on the downregulation of nestin immunoreactivity in progenitor cells. Opsin-positive rods could be reverted to an opsin-negative state initially, but became unresponsive to CNTF later. No compensatory increase in the number of other cell types was observed. Application of neutralizing antibodies against CNTF revealed that rod development was partially blocked by an endogenous CNTF-like molecule in control cultures. Our results suggest that CNTF can act as a specific negative regulator of rod differentiation. Its action on photoreceptor precursor cells could serve to synchronize the maturation of photoreceptors, which are born over an extended period of time. Together with other stimulatory signals, CNTF may thus control the temporally and numerically correct integration of photoreceptors into the retinal network.
In this study we describe a large-scale screening cell ELISA protocol which is suitable for the characterization of exogenic factor effects in mixed central nervous system (CNS) culture. The main novelty of the assay is that it permits the measurement of cellular responses in populations comprising as little as 2-4% of the total cell number. For standardization of the assay, we employed antibodies against opsin and microtubule-associated protein (MAP2) which label distinct retinal cell classes. Embryonic chick retinal neurons were grown in microtiter plates and directly processed for detection of antibody binding on the same plate. Binding of the antibodies was saturable and the ELISA signal was proportional to the number of immunoreactive cells comprising 2-4% and 16% of the total cell number with opsin and MAP2 antibodies, respectively. A minimum of 2000 opsin-positive cells could be reliably determined. Using our cell ELISA protocol, we demonstrate a developmental increase of both cell markers which reflected an increase in the number of opsin-positive cells but an enhanced expression per cell in the case of MAP2. We also show that growth-promoting activity-the presumed chick ciliary neurotrophic factor (CNTF)-stimulated the expression of opsin in retinal cultures (EC50; 2.3 pM) and that a corresponding activity is specifically expressed in the developing retina. Our results show that the cell ELISA protocol allows the rapid screening for distinct, low-percentage cell populations responding to exogenous factors in mixed CNS cultures.