The RhoGAP domain-containing protein, Porf-2, inhibits proliferation and enhances apoptosis in neural stem cells

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Abstract

Neural stem cells (NSCs) are essential to developing and mature CNS. They shape the structural and functional layouts of the brain in developing CNS and continue to proliferate, generating new neurons in several adult brain regions. Preoptic regulatory factor-2 (Porf-2), a RhoGAP domain-containing protein expressed in CNS, has a role in gender-related brain development and function. Porf-2 expression was knocked down in C17.2, a mouse cerebellar multipotent cell line. This increased proliferation and decreased drug-induced apoptosis without affecting cell type distribution following differentiation induction. It lowered levels of cyclin kinase inhibitor p21, affected G1 to S phase cell cycle transition; partially blocked the elevation in p53 transcriptional activity, p21 and Bcl-2-associated X protein (Bax) levels caused by bleomycin, but had no influence on enhancement of Bax in response to staurosporine. Thus Porf-2 may inhibit NSC proliferation by enhancing p21 protein levels followed by G1 phase arrest; it plays pro-apoptotic roles in response to drug treatment through both p53 transcription-dependent and independent pathways. This is consistent with categorization of Porf-2 as a functional RhoGAP in CNS.

Introduction

NSCs are undifferentiated cells that divide symmetrically to generate identical cells, and asymmetrically to produce progenitor cells giving rise to different cell types such as neurons and glia (Kriegstein and Alvarez-Buylla, 2009). NSCs shape the structural and functional layout of the brain in the developing CNS and continue to proliferate and generate new neurons in several areas of adult brain including SVZ and DG (Kokovay et al., 2008). Their fate plays a role in cortical architecture and expansion (Kriegstein et al., 2006) and the precise control of cell division and survival and cell cycle exit during development is critical to achieve the appropriate organization and volume of regions of the CNS, including the hippocampus, cerebral cortex and cerebellum (Kriegstein et al., 2006, Sillitoe and Joyner, 2007, Joseph and Hermanson, 2010). Changes in cell cycle length can influence development and physiopathology in the nervous system (Salomoni and Calegari, 2010).

P53, p21 and Bax play decisive roles in determining the fate of NSCs, influencing the balance of stem cell maintenance with proliferation, differentiation and apoptosis (Joseph and Hermanson, 2010). Cell division is dependent on cyclin-dependent kinases and their inhibitors and one of these, p21, is critical for the maintenance of stem cell renewal. P53, on the other hand, is a proapoptotic protein that negatively regulates self-renewal. Apoptosis or naturally occurring cell death plays an essential role in the developing nervous system. Over 50% of neurons that are generated undergo apoptosis during a restricted time window that varies from region to region (Oppenheim, 1991). Bax is a pro-death gene of the Bcl-2 family that is singularly important for apoptosis in the CNS (Forger, 2009).

NSCs and regulation of apoptosis may play a role in developmental disorders, learning and memory (Kriegstein et al., 2006), and a wide array of diseases of the CNS such as depression, neuroinflammation, epilepsy, cancer and neurodegenerative disorders, including Parkinson's, Alzheimer's and Huntington's (Grote and Hannan, 2007; Kriegstein and Alvarez-Bullya, 2009; Clarke, 2004, Zaidi et al., 2009, Yadirgi and Marino, 2009). Native or modified NSCs from spatially specific regions may become part of the therapeutic strategy for these disorders, as well as CNS injury due to autoimmune destruction, trauma or stroke (Kriegstein and Alvarez-Bullya, 2009; Daniela et al., 2007, Nandoe Tewarie et al., 2009, Sharp and Keirstead, 2009). C17.2 cells have been shown to integrate into the CNS and differentiate into neurons and glia (Snyder et al., 2004). Although these cells have shown therapeutic promise in several models of disease including spinal cord injury (Teng et al., 2002), cortical and cerebellar defects (Snyder et al., 2004) and Parkinson's (Ourednik et al., 2002), attempts to repair peripheral nerves can result in a high rate of tumor formation (Johnson et al., 2008). Determination of the mechanisms that regulate NSC growth may lead to better therapeutic strategies such as targeting specific genes or pathways in NSC in situ to increase or decrease self-renewal (Yadirgi and Marino, 2009).

Preoptic regulatory factor-2 (Porf-2) is a growth regulator found in the CNS. Porf-2 genetic loci are detected in a wide range of species: rat, mouse, human, pig, sheep, cow, chicken, and zebrafish (Nowak, 2003). The mouse and human homologues are D15Wsu169e and KIAA1688. Porf-2 is most highly expressed during neonatal and early postnatal life in rat hypothalamus (Nowak et al., 1999) but also in hippocampus, cerebral cortex, anterior pituitary and cerebellum. Age, gender, hormonal status, and brain region regulate expression (Nowak, 2003, Nowak et al., 1999, Nowak and Gore, 1999), and sexually dimorphic expression is observed in rat hypothalamus (POA-AH) during the perinatal period (Nowak and Gore, 1999).

Porf-2 is also expressed in peripheral tissues characterized by rapid cell division including skin, dividing testicular germ cells and placental growth cone (Nowak, 2003). Porf-2 contains a RhoGAP domain (Peck et al., 2002). Rho GTPases belong to Ras related small G protein family including Rho, Rac and Cdc42 that act as molecular switches (Etienne-Manneville and Hall, 2002). They have been implicated in tumor growth and invasion (Evers et al., 2000, Frame and Brunton, 2002). The GAPs (GTPase-activating proteins) enhance the intrinsic GTPase activity of Rho GTPases leading to the GDP-bound inactive state (Tcherkezian and Lamarche-Vane, 2007). Some Rho GAPs regulate cell proliferation and apoptosis (Nagaraja and Kandpal, 2004, Modarressi et al., 2004, Ching et al., 2003).

Under certain conditions Porf-2 inhibits growth of NIH3T3 cells (Nowak and Gilham, 2004); we reasoned that Porf-2 might display tumor suppressor function in NSCs. Expression of Porf-2 was knocked down by Porf-2 shRNA and NSC proliferation, apoptosis and differentiation were examined. Porf-2 inhibited cell proliferation, increasing p21 expression followed by G1 phase cell cycle arrest, and enhanced drug-induced apoptosis through both p53 transcription-dependent and independent pathways.

Section snippets

Creating a Porf-2 knockdown neural stem cell line

In C17.2 cells transduced by five different Porf-2 shRNAs, the relative expression level of Porf-2 mRNA was 28.5%, 81.7%, 52.9%, 61.7%, and 56.4% respectively, versus control cells (Fig. 1A). The reduction of Porf-2 mRNA level was statistically significant in four transduced cell lines, including 184-C17.2, 186-C17.2, 187-C17.2 and 188-C17.2. The 184-C17.2 clone, exhibited the highest reduction of Porf-2 mRNA and was chosen to confirm reduction of Porf-2 at the protein level. An immunoblot

Discussion

In the present study, the growth regulatory function of Porf-2, a RhoGAP domain containing protein, was examined in C17.2. Porf-2 was found to inhibit the cell proliferation of C17.2 and this inhibition was greater when serum supplementation was maximal. This result is consistent with previous data that Porf-2 suppressed cell growth of NIH3T3 cells under favorable conditions (Nowak and Gilham, 2004). The small but significant difference in cell metabolism seen with the Alamar blue assay was not

Cells

C17.2 cells were used to study the function of Porf-2 in NSCs. They are stable, multipotent NSCs which can differentiate into neurons, astrocytes, and glial precursors in vitro. C17.2 cells were originally derived from neonatal mouse cerebellar external granular layer and immortalized by introduction of v-myc (Ryder et al., 1990). The gender of the mice was not reported. C17.2 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Sigma-Aldrich Corp., St. Louis, MO) containing 4 mM 

Disclosure statement

The authors have nothing to disclose.

Acknowledgments

A more detailed account of this work is published as the dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy by SM. We thank Drs. Robert Colvin, Xiaozhuo Chen and Allan Showalter for their many helpful suggestions and comments during the completion of that dissertation. We thank Zhenchao Wang for help to SM with PCR assays and primer design and cell culture techniques. We thank Dr. Karen Coschigano for use of the real time PCR thermal cycler

References (56)

  • T. Miyashita et al.

    Overexpression of the Bcl-2 protein increases the half-life of p21Bax

    J. Biol. Chem.

    (1995)
  • G.M. Nagaraja et al.

    Chromosome 13q12 encoded Rho GTPase activating protein suppresses growth of breast carcinoma cells, and yeast two-hybrid screen shows its interaction with several proteins

    Biochem. Biophys. Res. Commun.

    (2004)
  • F.V. Nowak

    Expression and characterization of the preoptic regulatory factor-1 and -2 peptides

    Regul. Pept.

    (2003)
  • J. Peck et al.

    Human RhoGAP domain-containing proteins: structure, function and evolutionary relationships

    FEBS Lett.

    (2002)
  • C. Rudolph et al.

    Determination of copy number of c-Myc protein per cell by quantitative Western blotting.

    Anal. Biochem.

    (1999)
  • P. Salomoni et al.

    Cell cycle control of mammalian neural stem cells: putting a speed limit on G1

    Trends Cell Biol.

    (2010)
  • J. Sharp et al.

    Stem cell-based cell replacement strategies for the central nervous system

    Neurosci. Lett.

    (2009)
  • F.C. Yang et al.

    Rac2 stimulates Akt activation affecting BAD/Bcl-XL expression while mediating survival and actin function in primary mast cells

    Immunity

    (2000)
  • J.M. Adams et al.

    The Bcl-2 protein family: arbiters of cell survival

    Science

    (1998)
  • S.A. Amundson et al.

    Roles for p53 in growth arrest and apoptosis: putting on the brakes after genotoxic stress

    Oncogene

    (1998)
  • M.F. Clarke

    Neurobiology: at the root of brain cancer

    Nature

    (2004)
  • P.S. Costello et al.

    The GTPase rho controls a p53-dependent survival checkpoint during thymopoiesis

    J. Exp. Med.

    (2000)
  • F. Daniela et al.

    The stem cells as a potential treatment for neurodegeneration

    Meth. Mol. Biol.

    (2007)
  • W.S. El-Deiry et al.

    WAF1/CIP1 is induced in p53-mediated G1 arrest and apoptosis

    Cancer Res.

    (1994)
  • S. Etienne-Manneville et al.

    Rho GTPases in cell biology

    Nature

    (2002)
  • N.G. Forger

    Control of cell number in the sexually dimorphic brain and spinal cord

    J. Neuroendocrinol.

    (2009)
  • Y. Geng et al.

    p53 transcription-dependent and -independent regulation of cerebellar neural precursor cell apoptosis

    J. Neuropathol. Exp. Neurol.

    (2007)
  • Y. Geng et al.

    Cytoplasmic p53 and activated Bax regulate p53-dependent, transcription-independent neural precursor cell apoptosis

    J. Histochem. Cytochem.

    (2010)
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