Extracellular control of cell size.

Both cell growth (cell mass increase) and progression through the cell division cycle are required for sustained cell proliferation. Proliferating cells in culture tend to double in mass before each division, but it is not known how growth and division rates are co-ordinated to ensure that cell size is maintained. The prevailing view is that coordination is achieved because cell growth is rate-limiting for cell-cycle progression. Here, we challenge this view. We have investigated the relationship between cell growth and cell-cycle progression in purified rat Schwann cells, using two extracellular signal proteins that are known to influence these cells. We find that glial growth factor (GGF) can stimulate cell-cycle progression without promoting cell growth. We have used this restricted action of GGF to show that, for cultured Schwann cells, cell growth rate alone does not determine the rate of cell-cycle progression and that cell size at division is variable and depends on the concentrations of extracellular signal proteins that stimulate cell-cycle progression, cell growth, or both.

Calcitonin gene-related peptide promotes Schwann cell proliferation.

Schwann cells in culture divide in response to defined mitogens such as PDGF and glial growth factor (GGF), but proliferation is greatly enhanced if agents such as forskolin, which increases Schwann cell intracellular cAMP, are added at the same time as PDGF or GGF (Davis, J. B., and P. Stroobant. 1990. J. Cell Biol. 110:1353-1360). The effect of forskolin is probably due to an increase in numbers of PDGF receptors (Weinmaster, G., and G. Lemke. 1990. EMBO (Eur. Mol. Biol. Organ.) J. 9:915-920. Neuropeptides and beta-adrenergic agonists have been reported to have no effect on potentiating the mitogenic response of either PDGF or GGF. We show that the neuropeptide calcitonin gene-related peptide (CGRP) increases Schwann cell cAMP levels, but the cells rapidly desensitize. We therefore stimulated the cells in pulsatile fashion to partly overcome the effects of desensitization and show that CGRP can synergize with PDGF to stimulate Schwann cell proliferation, and that CGRP is as effective as forskolin in the pulsatile regime. CGRP is a good substrate for the neutral endopeptidase 24.11. Schwann cells in vivo have this protease on their surface, so the action of CGRP could be terminated by this enzyme and desensitization prevented. We therefore suggest that CGRP may play an important role in stimulating Schwann cell proliferation by regulating the response of mitogenic factors such as PDGF.

Cultured Schwann cells constitutively express the myelin protein P0.

It is widely thought that mammalian Schwann cells do not express Po, the major glycoprotein in peripheral myelin, unless they are induced to do so by axonal signals that can be mimicked by agents that trigger cAMP signaling pathways. In contrast, we find that cultured Schwann cells make large amounts of Po without the addition of any axonal-like signal, provided they have not been exposed to serum during the culture process. We also report that glial growth factor/neuregulin inhibits this constitutive Po expression. Myelin basic protein is regulated in a similar way. We suggest that expression of Po by Schwann cells before the onset of myelination may be prevented by inhibitory signals within the nerve, rather than by the absence of a positive signal from axons.

Evidence that axon-derived neuregulin promotes oligodendrocyte survival in the developing rat optic nerve.

It was previously shown that newly formed oligodendrocytes depend on axons for their survival, but the nature of the axon-derived survival signal(s) remained unknown. We show here that neuregulin (NRG) supports the survival of purified oligodendrocytes and aged oligodendrocyte precursor cells (OPCs) but not of young OPCs. We demonstrate that axons promote the survival of purified oligodendrocytes and that this effect is inhibited if NRG is neutralized. In the developing rat optic nerve, we provide evidence that delivery of NRG decreases both normal oligodendrocyte death and the extra oligodendrocyte death induced by nerve transection, whereas neutralization of endogenous NRG increases the normal death. These results suggest that NRG is an axon-associated survival signal for developing oligodendrocytes.

Programmed cell death by default in embryonic cells, fibroblasts, and cancer cells.

We recently proposed that most mammalian cells constitutively express all of the proteins required to undergo programmed cell death (PCD) and undergo PCD unless continuously signaled by other cells not to. Although some cells have been shown to work this way, the vast majority of cell types remain to be tested. Here we tested purified fibroblasts isolated from developing or adult rat sciatic nerve, a mixture of cell types isolated from normal or p53-null mouse embryos, an immortalized rat fibroblast cell line, and a number of cancer cell lines. We found the following: 1) All of these cells undergo PCD when cultured at low cell density in the absence of serum and exogenous signaling molecules but can be rescued by serum or specific growth factors, suggesting that they need extracellular signals to avoid PCD. (2) The mixed cell types dissociated from normal mouse embryos can only support one another's survival in culture if they are in aggregates, suggesting that cell survival in embryos may depend on short-range signals. (3) Some cancer cells secrete factors that support their own survival. (4) The survival requirements of a human leukemia cell line change when the cells differentiate. (5) All of the cells studied can undergo PCD in the presence of cycloheximide, suggesting that they constitutively express all of the protein components required to execute the death program.

Evidence that CGRP and cAMP increase transcription of AChR alpha-subunit gene, but not of other subunit genes.

Calcitonin gene-related peptide (CGRP) is present in embryonic chick motoneurons and their terminals during myogenesis. We have studied the effect of CGRP on the expression of mRNA encoding the four subunits (alpha, beta, gamma, delta) of ACh receptors in cultured myotubes derived from embryonic chicks. Northern blot analysis showed that treatment with 10(-7) M CGRP caused an increase in ACh receptor alpha-subunit mRNA expression but did not affect the expression of beta-, gamma-, or delta-subunit mRNAs. In addition, CGRP treatment caused an increase in the expression of unspliced alpha-subunit RNA, suggesting that CGRP increases transcription of the alpha-subunit gene. The effect of CGRP on alpha-subunit gene transcription was mimicked by forskolin, and both CGRP and forskolin increased the levels of intracellular cAMP. We infer that the effect of CGRP on alpha-subunit gene transcription is likely to be mediated by the CGRP-induced rise in intracellular cAMP.

Chick myotubes in culture express high-affinity receptors for calcitonin gene-related peptide.

Calcitonin gene-related peptide (CGRP) is a 37-amino acid neuropeptide that is expressed by many neurons of the vertebrate nervous system, including motoneurons of many species. It has been detected immunohistochemically in both cell bodies and motor terminals of motoneurons, suggesting that it may play a role at the neuromuscular junction. In support of this idea, CGRP has been shown to produce a variety of effects on cultured myotubes and muscle explants, including elevation of cAMP levels, increase in cell-surface acetylcholine receptor (AChR) numbers, increase in AChR alpha-subunit mRNA transcript levels, alterations in contractile responses, alterations in the physiological properties of AChRs, and inhibition of insulin-induced changes in glycogen metabolism. CGRP binding sites have been detected in many tissues, but have not yet been demonstrated directly on muscle cells. Here we report that chick myotubes in culture express high-affinity binding sites for CGRP (Kd approximately 2-4 x 10(-10) M). In view of the known biological effects of CGRP on myotubes, we believe that these binding sites represent CGRP receptors. They are uniformly distributed over the surface of myotubes, and we have found no evidence of clustering in culture, in contrast to AChRs. We have found no evidence for more than one class of receptors.

Onset of CGRP expression and its restriction to a subset of spinal motor neuron pools in the chick embryo is not affected by treatment with curare.

Calcitonin gene-related peptide (CGRP) is expressed in and defines a subset of motor neuron pools in the lumbar spinal cord of the chick embryo. The onset of CGRP expression in individual pools coincides with both the period of the initial innervation of the leg and the beginning of naturally occurring cell death in the lumbar motor column. Administration of neuromuscular blocking agents at this time results in a striking reduction of normal motor neuron loss. It has been reported that such treatment also results in the abolition of CGRP expression at later stages of development. In this study, we have examined the effect of curare treatment on CGRP expression in motor neurons earlier in their development. We find that, in contrast to the effects reported at later stages, inhibition of neuromuscular activity does not affect either the onset of CGRP expression or its restriction to a subset of motor neuron pools. This demonstrates that the control of the onset of CGRP expression is unlikely to be linked to processes which are regulated by neuromuscular transmission including naturally-occurring cell death.