Reg-2 is a motoneuron neurotrophic factor and a signalling intermediate in the CNTF survival pathway

Abstract

Cytokines that are related to ciliary neurotrophic factor (CNTF) are physiologically important survival factors for motoneurons, but the mechanisms by which they prevent neuronal cell death remain unknown. Reg-2/PAP I (pancreatitis-associated protein I), referred to here as Reg-2, is a secreted protein whose expression in motoneurons during development is dependent on cytokines. Here we show that CNTF-related cytokines induce Reg-2 expression in cultured motoneurons. Purified Reg-2 can itself act as an autocrine/paracrine neurotrophic factor for a subpopulation of motoneurons, by stimulating a survival pathway involving phosphatidylinositol-3-kinase, Akt kinase and NF-κB. Blocking Reg-2 expression in motoneurons using Reg-2 antisense adenovirus specifically abrogates the survival effect of CNTF on cultured motoneurons, indicating that Reg-2 expression is a necessary step in the CNTF survival pathway. Reg-2 shows a unique pattern of expression in late embryonic spinal cord: it is progressively upregulated in individual motoneurons on a cell-by-cell basis, indicating that only a fraction of motoneurons in a given motor pool may be exposed to cytokines. Thus, Reg-2 is a neurotrophic factor for motoneurons, and is itself an obligatory intermediate in the survival signalling pathway of CNTF-related cytokines.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Expression of Reg-2 in motoneurons is tightly regulated by CNTF-related cytokines.
Figure 2: Reg-2 is an autocrine/paracrine motoneuron survival factor.
Figure 3: Reg-2 signals for survival through PI(3)K/Akt kinase and NF-κB.
Figure 4: CNTF survival signalling requires Reg-2.
Figure 5: Fraction of motoneurons expressing Reg-2 increases with time during the period of naturally occurring motoneuron cell death.
Figure 6: Reg-2 expression in vivo is controlled on a cell-by-cell basis.

References

  1. 1

    Henderson, C. E. Role of neurotrophic factors in neuronal development. Curr. Opin. Neurobiol. 6, 64–70 ( 1996).

  2. 2

    Stahl, N. et al. Cross-linking identifies leukemia inhibitory factor-binding protein as a ciliary neurotrophic factor receptor component. J. Biol. Chem. 268, 7628–7631 ( 1993).

  3. 3

    Heinrich, P. C., Behrmann, I., Muller-Newen, G., Schaper, F. & Graeve, L. Interleukin-6-type cytokine signalling through the gp130/Jak/STAT pathway. Biochem. J. 334, 297–314 ( 1998).

  4. 4

    Henderson, C. E. et al. GDNF: a potent survival factor for motoneurons present in peripheral nerve and muscle. Science 266, 1062–1064 (1994).

  5. 5

    Arakawa, Y., Sendtner, M. & Thoenen, H. Survival effect of ciliary neurotrophic factor (CNTF) on chick embryonic motoneurons in culture: comparison with other neurotrophic factors and cytokines. J. Neurosci. 10, 3507–3515 (1990).

  6. 6

    Sendtner, M., Kreutzberg, G. W. & Thoenen, H. Ciliary neurotrophic factor prevents the degeneration of motor neurons after axotomy. Nature 345, 440–441 (1990).

  7. 7

    Pennica, D. et al. Cardiotrophin-1, a cytokine present in embryonic muscle, supports long-term survival of spinal motoneurons. Neuron 17 , 63–74 (1996).

  8. 8

    Sendtner, M. et al. Ciliary neurotrophic factor prevents degeneration of motor neurons in mouse mutant progressive motor neuronopathy. Nature 358, 502–504 ( 1992).

  9. 9

    Sendtner, M. Gene therapy for motor neuron disease. Nature Med. 3, 380–381 (1997).

  10. 10

    Sagot, Y., Tan, S. A., Hammang, J. P., Aebischer, P. & Kato, A. C. GDNF slows loss of motoneurons but not axonal degeneration or premature death of pmn/pmn mice . J. Neurosci. 16, 2335– 2341 (1996).

  11. 11

    Mitsumoto, H. et al. Arrest of motor neuron disease in wobbler mice cotreated with CNTF and BDNF. Science 265, 1107– 1110 (1994).

  12. 12

    Haase, G. et al. Gene therapy of murine motor neuron disease using adenoviral vectors for neurotrophic factors. Nature Med. 3, 429–436 (1997).

  13. 13

    Okamoto, H. The Reg gene family and Reg proteins: with special attention to the regeneration of pancreatic β-cells. J. Hepatobiliary Pancreat. Surg. 6, 254–262 (1999).

  14. 14

    Iovanna, J., Orelle, B., Keim, V. & Dagorn, J. C. Messenger RNA sequence and expression of rat pancreatitis-associated protein, a lectin-related protein overexpressed during acute experimental pancreatitis. J. Biol. Chem. 266, 24664–24669 (1991).

  15. 15

    Orelle, B., Keim, V., Masciotra, L., Dagorn, J. C. & Iovanna, J. L. Human pancreatitis-associated protein. Messenger RNA cloning and expression in pancreatic diseases. J. Clin. Invest. 90, 2284–2291 (1992).

  16. 16

    Ortiz, E. M. et al. The pancreatitis-associated protein is induced by free radicals in AR4-2J cells and confers cell resistance to apoptosis. Gastroenterology 114, 808–816 ( 1998).

  17. 17

    de la Monte, S. M., Ozturk, M. & Wands, J. R. Enhanced expression of an exocrine pancreatic protein in Alzheimer's disease and the developing human brain. J. Clin. Invest. 86, 1004–1013 (1990).

  18. 18

    Livesey, F. J. et al. A Schwann cell mitogen accompanying regeneration of motor neurons. Nature 390, 614– 618 (1997).

  19. 19

    Dusetti, N. J., Ortiz, E. M., Mallo, G. V., Dagorn, J. C. & Iovanna, J. L. Pancreatitis-associated protein I (PAP I), an acute phase protein induced by cytokines. Identification of two functional interleukin-6 response elements in the rat PAP I promoter region. J. Biol. Chem. 270, 22417– 22421 (1995).

  20. 20

    Arce, V. et al. Cardiotrophin-1 requires LIFRβ to promote survival of mouse motoneurons purified by a novel technique. J. Neurosci. Res. 55, 119–126 (1999).

  21. 21

    Kobayashi, S. et al. Identification of a receptor for reg (regenerating gene) protein, a pancreatic beta-cell regeneration factor. J. Biol. Chem. 275, 10723–10726 (2000).

  22. 22

    Middleton, G. et al. Cytokine-induced nuclear factor kappa B activation promotes the survival of developing neurons. J. Cell Biol. 148 , 325–332 (2000).

  23. 23

    Romashkova, J. A. & Makarov, S. S. NF-κB is a target of AKT in anti-apoptotic PDGF signalling. Nature 401, 86–90 (1999).

  24. 24

    Yamamoto, Y. & Henderson, C. E. Patterns of programmed cell death in populations of developing spinal motoneurons in chicken, mouse, and rat. Dev. Biol. 214, 60– 71 (1999).

  25. 25

    MacLennan, A. J. et al. Immunohistochemical localization of ciliary neurotrophic factor receptor alpha expression in the rat nervous system. J. Neurosci. 16, 621–630 ( 1996).

  26. 26

    Li, M., Sendtner, M. & Smith, A. Essential function of LIF receptor in motor neurons . Nature 378, 724–727 (1995).

  27. 27

    deLapeyriere, O. & Henderson, C. E. Motoneuron differentiation, survival and synaptogenesis. Curr. Opin. Genet. Dev. 7, 642–650 ( 1997).

  28. 28

    Oppenheim, R. W. Neurotrophic survival molecules for motoneurons: an embarrassment of riches . Neuron 17, 195–197 (1996).

  29. 29

    Davies, A. M. & Wright, E. M. Neurotrophic factors. Neurotrophin autocrine loops. Curr. Biol. 5, 723–726 (1995).

  30. 30

    Acheson, A. et al. A BDNF autocrine loop in adult sensory neurons prevents cell death. Nature 374, 450– 453 (1995).

  31. 31

    Maina, F. et al. Multiple roles for hepatocyte growth factor in sympathetic neuron development. Neuron 20, 835– 846 (1998).

  32. 32

    Ernfors, P., Merlio, J.-P. & Persson, H. Cells expressing mRNA for neurotrophins and their receptors during embryonic rat development. Eur. J. Neurosci. 4, 1140–1158 (1992).

  33. 33

    Henderson, C. E. et al. Neurotrophins promote motor neuron survival and are present in embryonic limb bud. Nature 363, 266– 270 (1993).

  34. 34

    Kahane, N., Shelton, D. L. & Kalcheim, C. Expression and regulation of brain-derived neurotrophic factor and neurotrophin-3 mRNAs in distinct avian motoneuron subsets. J. Neurobiol. 29, 277–292 (1996).

  35. 35

    Garcès, A., Nishimune, H., Philippe, J. M., Pettmann, B. & deLapeyrière, O. FGF9: a motoneuron survival factor expressed by medial thoracic and sacral motoneurons. J. Neurosci. Res. 60, 1– 9 (2000).

  36. 36

    Pettmann, B. & Henderson, C. E. Neuronal cell death. Neuron 20, 633–647 (1998).

  37. 37

    Wiese, S. et al. The anti-apoptotic protein ITA is essential for NGF-mediated survival of embryonic chick neurons. Nature Neurosci. 2, 978–983 (1999).

  38. 38

    Raoul, C., Henderson, C. E. & Pettmann, B. Programmed cell death of embryonic motoneurons triggered through the Fas death receptor. J. Cell Biol. 147, 1049–1062 (1999).

  39. 39

    Tanabe, Y. & Jessell, T. M. Diversity and pattern in the developing spinal cord. Science 274, 1115–1123 (1996).

  40. 40

    Harris, A. J. & McCaig, C. D. Motoneuron death and motor unit size during embryonic development of the rat. J. Neurosci. 4, 13–24 ( 1984).

  41. 41

    Nakashima, K. et al. Developmental requirement of gp130 signaling in neuronal survival and astrocyte differentiation. J. Neurosci. 19, 5429–5434 (1999).

  42. 42

    Mettling, C. et al. Survival of newly postmitotic motoneurons is transiently independent of exogenous trophic support. J. Neurosci. 15, 3128–3137 (1995).

  43. 43

    Gould, T. W., Burek, M. J., Sosnowski, J. M., Prevette, D. & Oppenheim, R. W. The spatial-temporal gradient of naturally occurring motoneuron death reflects the time of prior exit from the cell cycle and position within the lateral motor column. Dev. Biol. 216, 611–621 (1999).

  44. 44

    McKay, S. E. et al. The expression of trkB and p75 and the role of BDNF in the developing neuromuscular system of the chick embryo. Development 122, 715–724 ( 1996).

  45. 45

    Stöckli, K. A. et al. Molecular cloning, expression and regional distribution of rat ciliary neurotrophic factor. Nature 342, 920–923 (1989).

  46. 46

    Sendtner, M. et al. Cryptic physiological trophic support of motoneurons by LIF revealed by double gene targeting of CNTF and LIF. Curr. Biol. 6, 686–694 ( 1996).

  47. 47

    DeChiara, T.M. et al. Mice lacking the CNTF receptor, unlike mice lacking CNTF, exhibit profound motor neuron deficits at birth. Cell 83, 313–322 (1995).

  48. 48

    Funakoshi, H. et al. Muscle-derived neurotrophin-4 as an activity-dependent trophic signal for adult motor neurons. Science 268, 1495–1499 (1995).

  49. 49

    Bödeker, H., Keim, V., Fiedler, F., Dagorn, J. C. & Iovanna, J. PAP I interacts with itself, PAP II, PAP III and lithostathine/regIa. Mol. Biol. Cell. Res. Comm. 2, 150–154 ( 1999).

  50. 50

    Foxwell, B. et al. Efficient adenoviral infection with IkappaB alpha reveals that macrophage tumor necrosis factor alpha production in rheumatoid arthritis is NF-κB dependent. Proc. Natl Acad. Sci. USA 95, 8211–8215 (1998).

Download references

Acknowledgements

We thank J. R. Sanes and members of U.382 for valuable discussions and critical review of the manuscript. We are grateful to S. P. Hunt for anti Reg-2 antibody; J. R. Sanes for anti β-gal antibody; T. Jessell for Isl1 and Chat plasmids; H. Bödeker for purification of Reg-2 protein and rat Reg-2 plasmid; D. Pennica for CT-1 null mutant mice; C. Lasserre for mouse Reg-2 plasmid; C. J. Woolf for adenovirus coding LacZ; A. Garcès and J. Livet for the WM-ISH technique for spinal cord; V. Arce for technical suggestions; R. Goetz for genotyping the LIF/CNTF knockout embryo; and C. Moretti for confocal microscopy techniques. This work was funded by the Institut National de la Santé et de la Recherche Médicale, Centre National de la Recherche Scientifique, Association Française contre les Myopathies (AFM), Institut pour la Recherche sur la Moelle Epinière (IRME), and European Commission BIO4 contract CT960433. H.N. was supported by JST Overseas Research Fellowship and a postdoctoral fellowship from NOVARTIS Foundation (Japan).

Author information

Correspondence to Christopher E. Henderson.

Rights and permissions

Reprints and Permissions

About this article

Further reading