Cell Cycle Inhibitors p21 and p16 Are Required for the Regulation of Schwann Cell Proliferation SUZANA ATANASOSKI, 1 DANIELLE BOLLER, 1 LUKAS DE VENTURA, 1 HEIDI KOEGEL, 1 MATTHIAS BOENTERT, 2 PETER YOUNG, 2 SABINE WERNER, 1 AND UELI SUTER 1 * 1 Department of Biology, Institute of Cell Biology, Swiss Federal Institute of Technology, ETH-H€ onggerberg, Zurich, Switzerland 2 Department of Neurology, University of M€ unster, M€ unster, Germany KEY WORDS peripheral nerves; glia; development; nerve injury; cyclin- dependent kinase inhibitors ABSTRACT Regulated cell proliferation is a crucial prerequisite for Schwann cells to achieve myelination in development and regeneration. In the present study, we have investigated the function of the cell cycle inhibitors p21 and p16 as potential regulators of Schwann cell proliferation, using p21- or p16-deficient mice. We report that both inhibitors are required for proper withdrawal of Schwann cells from the cell cycle during development and following injury. Postnatal Schwann cells express p21 exclusively in the cytoplasm, first detectable at postnatal day 7. This cytoplas- mic p21 expression is necessary for proper Schwann cell proliferation control in the late development of peripheral nerves. After axonal damage, p21 is found in Schwann cell nuclei during the initiation of the proliferation period. This stage is critically regulated by p21, since loss of p21 leads to a strong increase in Schwann cell proliferation. Unex- pectedly, p21 levels are upregulated in this phase suggest- ing that the role of p21 may be more complex than purely inhibitory for the Schwann cell cycle. However, inhibition of Schwann cell proliferation is the overriding crucial function of p21 and p16 in peripheral nerves as revealed by the con- sequences of loss-of-function in development and after in- jury. Different mechanisms appear to underlie the inhibi- tory function, depending on whether p21 is cytoplasmic or nuclear. V V C 2005 Wiley-Liss, Inc. INTRODUCTION The development of Schwann cells requires the precise integration of controlled proliferation and regulated cell cycle exit. These processes are accompanied by morpho- logical changes that culminate in the specialized nature of mature myelinating and nonmyelinating Schwann cells in adult nerves (Lobsiger et al., 2002). Impaired control of these fundamental processes in Schwann cell biology is associated with diseases and pathological states including inherited peripheral neuropathies (Atanasoski et al., 2002; Suter and Scherer, 2003), peri- pheral nerve tumors (Riccardi, 1991), and peripheral neuropathies secondary to diabetes, cancer chemothera- peutic agents, or toxins (Berger and Schaumburg, 1995). Schwann cell proliferation reaches a peak around birth and gradually decreases when the cells begin to differenti- ate (Scherer et al., 1994; Mirsky et al., 2002). Following nerve injury, axons distal to the site of injury degenerate. As a consequence, Schwann cells dedifferentiate, accompa- nied by a wave of proliferation (Scherer et al., 1994). We and other investigators have shown that injury-induced Schwann cell proliferation requires cyclin D1 (Kim et al., 2000; Atanasoski et al., 2001), while developmentally re- gulated proliferation does not. Thus, the molecular con- trols of Schwann cell proliferation during nerve develop- ment and following injury differ significantly. Cell proliferation is controlled by the activation of cyclin-dependent kinases (cdks), which associate with cyclins D and E, and drive cells through the G1 phase of the cell cycle (Sherr, 1993, 1994; Roberts, 1999). Two classes of cdk inhibitors modulate the activity of cdk– cyclin complexes. The Cip/Kip family affects cyclin E– cdk2 complexes, while the Ink family targets preferen- tially cyclin D–cdk4/6 (Ortega et al., 2002; Sherr and Roberts, 1999). Biochemical activities of the inhibitors and their expression patterns during development impli- cate these proteins in the regulation of cell proliferation and cell differentiation (Zindy et al., 1997). There is a relative scarcity of available data on the reg- ulation and function of cell cycle inhibitors in vivo. Peri- pheral nerve development and injury-induced Schwann cell proliferation provide excellent paradigms to assess this issue in different but related settings. In the present study, we have analyzed the functional roles of p21 as a representative of the Cip/Kip family and p16 for the Ink family. We found that p21 appears first in the cytoplasm of Schwann cells during late postnatal development when most cells have already ceased dividing. This expression and cellular localization of p21 is maintained into adult- hood. After nerve injury, however, we found p21 mainly in nuclei of dedifferentiated Schwann cells. Consistent with the expression data, p21-deficient Schwann cells divide normally in early nerve development, but cytoplas- mic p21 is necessary for proper growth arrest in later Grant sponsor: Kommission Innovative Forschung of the University of M€ unster; Grant number: YO103231; Grant sponsor: Swiss National Science Foundation; Grant sponsor: NCCR Neural Plasticity and Repair. *Correspondence to: Ueli Suter, Institute of Cell Biology, ETH-H€ onggerberg, CH- 8093 Z€ urich, Switzerland. E-mail: usuter@cell.biol.ethz.ch Received 27 April 2005; Accepted 29 June 2005 DOI 10.1002/glia.20263 Published online 3 October 2005 in Wiley InterScience (www.interscience. wiley.com). V V C 2005 Wiley-Liss, Inc. GLIA 53:147–157 (2006)