NGF influences multiple functions depending on the cellular context and the specific receptors expressed and activated. The effects of NGF on neurons are well characterized and range from maintaining survival and differentiation via TrkA, to triggering apoptosis via p75NTR
). However, the effects on of NGF astrocytes have not been extensively characterized. We previously demonstrated that NGF causes an attenuation of astrocyte proliferation mediated by p75NTR
(Cragnolini et al., 2009
). Although we have used NGF in these studies to activate p75NTR
on the astrocytes, it is not clear what the endogenous ligand might be that activates p75NTR
to regulate astrocyte proliferation in vivo. Since proNGF is a more potent and selective ligand for p75NTR
, this proneurotrophin may be the actual in vivo ligand. The goal of this study was to investigate the cellular mechanisms responsible for growth arrest of astrocytes induced by NGF. We demonstrate that NGF inhibits the synthesis of specific cyclins and their interaction with CDKs, and prevents degradation of specific CIKs.
The cyclic changes in the expression of cyclins and their association with cdks are indispensable for cell cycle progression in all multicellular eukaryotes (Satyanarayana and Kaldis, 2009
). We observed that NGF attenuated the EGF-induced expression of cyclin D1, a key cyclin for the progression of the cell cycle through the G1/S phase. NGF treatment reduced, but did not completely prevent cyclin D and E induction by EGF, consistent with our previous study demonstrating that NGF attenuated, but did not completely inhibit, astrocyte proliferation (Cragnolini et al., 2009
). Although NGF did not diminish Cdk4 levels, the association with cyclin D1 was greatly reduced. When astrocytes were treated with EGF we detected an increase in both cyclin E expression and accumulation in the nucleus, consistent with its role to target nuclear substrates. NGF attenuated the cyclin E expression and inhibited its translocation to the nucleus. These findings suggest that the inhibition of cyclin expression and the exclusion of cyclin E from the nucleus may be mechanisms that contribute to the inhibition of cell cycle progression by NGF.
The activities and functions of Cdk/cyclin complexes are regulated by two families of Cdk inhibitors, the INK4 family that bind to Cdk4 and Cdk6 and prevent D-type cyclin activity and the Cip/Kip family that inhibits Cdk2/cyclinE (Toyoshima and Hunter, 1994
); (Aprelikova et al., 1995
; O'Connor, 1997
). The presence of NGF attenuated the degradation of p15INK and p27kip1, which is another mechanism by which NGF may arrest cell cycle progression. This is consistent with a previous study in neuroblastoma, in which an NGF-induced decrease in cell number was accompanied by an increase in p27kip1 levels (Woo et al., 2004
). The increased levels of these CDK inhibitors following NGF treatment could result from insufficient capacity of the cell to degrade these proteins. The post-translational regulation of cell cycle proteins can be predominantly achieved by two types of protein modification, phosphorylation and ubiquitination. In astrocytes, inhibition of the ubiquitin-proteasome system by lactasystin has been shown to inhibit cell cycle progression and proliferation through modifying cell cycle related proteins (Ren et al., 2009
). NGF affected the tightly controlled regulation of p27kip1 by inhibiting its ubiquitination. Other ubiquitinated proteins can also be positively or negatively influenced by NGF. For example, the binding of NGF to TrkA induces the internalization and ubiquitination of the receptor (Wooten and Geetha, 2006
) and in PC12 cells NGF induces differentiation by repressing the ubiquitination of T-cadherin (Bai et al., 2007
). In sympathetic neurons, NGF blocks the ubiquitin dependent degradation of Ret protein, increasing the levels of this protein and enhancing growth (Pierchala et al., 2007
). These data suggest that NGF can affect the levels of certain proteins either by increasing or decreasing their catabolism, depending on the cell context.
We previously showed that NGF induces the expression of p75NTR
in astrocytes, which may result in differences in signaling in response to NGF throughout the cell cycle. The p75NTR
lacks intrinsic catalytic activity and signaling depends on the ability to recruit specific cytoplasmic proteins that interact with its intracellular domain and trigger different signaling pathways. Several p75NTR
-binding proteins have been identified that are implicated in regulating cell proliferation. In particular, SC1, acts as a transcriptional repressor of the promitotic gene, cyclin E
, upon NGF treatment and thus blocks DNA replication (Chittka et al., 2004
). Interestingly, a recent publication demonstrated that the NGF promotes the association of the p75NTR
intracellular domain with the cyclin E promoter region and can modulate cylin E1 levels in PC12 cells (Parkhurst et al., 2010
). Additional p75NTR
-interacting proteins such as RIP2, Bex1, and Sall2, have also been implicated as regulators of the cell cycle (Khursigara et al. 2001
; Vilar et al, 2006
; Pincheira et al., 2009
The cell cycle arrest induced by NGF has been associated with the initiation of differentiation. PC12 cells respond to NGF by differentiating, and cells accumulate in the G1 phase of the cell cycle (van Grunsven et al., 1996
). In C6 astrocytoma cells NGF inhibited proliferation and induced morphological changes, including the formation of growth cones, outgrowth of processes and cellular hypertrophy, indicating that exogenous NGF stimulated differentiation and inhibited proliferation of these cells (Watanabe et al., 1999
The precise biological significance of the growth arrest and its role in reactive astrogliosis has not been elucidated yet, however it might have several consequences for nervous system function, especially after an injury. Consistent with the possibility that NGF modulates aspects of gliosis, Cirillo and colleges (2009)
showed that NGF reduced the reactive astrocytosis associated with neuropathic pain. NGF also was able to restore glial and neuronal amino acid transporters (Cirillo et al., 2011
). The potential outcome of NGF treatment on astrocytes may depend on the neurotrophin receptors expressed in a particular cellular context. Our in vitro experiments demonstrated that a scratch lesion increased levels of p75NTR
in the astrocytes, and that NGF prevented the EGF-induced BrdU incorporation in astrocytes after a scratch lesion, consistent with the idea that activation of p75NTR
may serve to attenuate gliosis after injury. In vivo, we have observed that p75NTR
is induced on astrocytes after seizures, although the specific role mediated by p75NTR
under these conditions remains unclear. Moreover, although we have used NGF to activate p75NTR
-mediated cell cycle arrest in these studies, many ligands can interact with this receptor, and although NGF levels are induced after injury, the endogenous ligand that may elicit these responses has not been elucidated.
Altogether our results reveal that profound changes in the cell cycle regulatory machinery can be mediated by the p75NTR, which signals astrocytes to withdraw from the cell cycle in response to NGF. During development, activation of p75NTR may participate in a regulatory mechanism for the cessation of proliferation prior to differentiation, and after injury the induction of p75NTR may serve to attenuate the extent of gliosis. The possibility that this neurotrophin receptor may regulate reactive gliosis in vivo after an injury or a neurodegenerative disease requires further investigation.