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I dont know whether this is of any interest.
Colostrinin-Driven Neurite Outgrowth Requires
p53 Activation in PC12 Cells
DISCUSSION
This study demonstrates for the first time that CLN causes PC12 cell proliferation
to cease, and induces morphological changes resembling cell differentiation.
The steps involved are p53 activation by phosphorylation, increased p21WAF1 expression
and cell cycle arrest in the G1 phase, followed by neurite outgrowth. The
CLN-induced neurite outgrowth was concentration-dependent, and apparently specific
to PC12 cells. PC12 cells respond to agonists with changes in gene expression
and undergo neural differentiation (Greene and Tischler, 1976; Levi et al., 1988;
Schwamborn et al., 2004; Vaudry et al., 2002; Yoo et al., 2004). The increase in the
percent of PC12 cells showing CLN-mediated neurite outgrowth and changes in
morphology resembled those for NGF-induced cell differentiation. We used NGF
as a positive control, because this is one of the best characterized agonists that induces
cell differentiation in PC12 cells (Levi et al., 1988; Vaudry et al., 2002). CLN,
like NGF, induces GAP-43 expression in PC12 cells during differentiation processes
(Costello et al., 1990; Jap Tjoen San et al., 1991; Przyborski and Cambray-Deakin,
1994; Ramakers et al., 1995). GAP-43 is a marker of neuronal differentiation whose
expression is restricted to the nervous system during development and regeneration
(for a review, see Benowitz and Routtenberg, 1997). Our immunochemical studies
showed that in CLN-differentiated cells GAP-43 was localized to the perinuclear
regions and cell extensions, consistent with studies showing that GAP-43 is primarily
found in growing neurite-like extensions and axons, where it primarily binds
membrane structures (Skene and Virag, 1989). Differentiation of PC12 cells by neurotrophins
(Mohiuddin et al., 1995), Neu differentiation factor (Vaskovsky et al.,
2000), IL-6 (Wu and Bradshaw, 1996), and IGF (Sumantran and Feldman, 1993)
associated cell cycle arrest involve the p53–p21WAF1 pathway; however, there are no
data showing that GAP-43 promoter is a transcriptional target for p53. Thus, GAP-
43 expression in CLN-treated cells may result from distant downstream events, and
be only indirectly dependent on p53–p21WAF1 pathway.
p53 exists in latent and antiproliferative forms that differ in their degree of posttranslational
modification, e.g., phosphorylation (Harris and Levine, 2005; Levine,
Cell Cycle Arrest and Neurite Outgrowth by Colostrinin 1135
1997) and their subcellular distribution (Shaulsky et al., 1991). Activated p53 alters
the transcription of genes, many of which regulate cell proliferation (Harris
and Levine, 2005; Levine, 1997; Liebermann et al., 1995) and are required for differentiation
(Aloni-Grinstein et al., 1993; Poluha et al., 1997). CLN-mediated inhibition
of cell proliferation via the p53–p21WAF1 pathway was predicted from the
accumulation of cells in the G1 phase of the cell cycle and lack of BrdU incorporation
into the DNA. We indeed observed an increase in p53ser15 levels that
accumulated in the nuclei of CLN-treated PC12 cells. These events occurred in
parallel with cell cycle arrest. Transfection of inhibitory oligonucleotides decreased
p53 below the detectable level over a 24-h time period. When these cells were
treated with CLN there was no p21WAF1 expression detectable, or arrest of cell
cycle observed. In independent studies, PC12 cells treated with p53 inhibitory
oligonucleotides continued through the cell cycle, confirming the dependence of
the NGF growth arrest signal on a p53 pathway (Hughes et al., 2000). This effect
appears to be specific to p53, because an oligonucleotide to luciferase did not alter
p53ser15 levels or expression of its target gene, p21WAF1 (el-Deiry et al., 1994).
In our experiments, PC12 cells transfected with inhibitory oligonucleotides specific
for p21WAF1 knocked out CLN-induced p21WAF1 expression, while p53 activation
was not altered. Both p53 and p21WAF1 inhibitory oligonucleotides significantly
decreased the percentage of cells showing neurite outgrowth in CLN- and
NGF-treated cultures. Thus, early CLN-induced molecular events closely resemble
those for NGF-induced ones, which are followed by G1 phase arrest and neurite
outgrowth.
CLN binds to the outer membrane of PC12 cells (Boldogh, 2001a, 2001b) and
induces the p53–p21WAF1 cascade, leading to cell cycle arrest and neurite outgrowth of
PC12 cells as we show in this study. This raises the possibility that CLN may interact
with the NGF receptor and utilize a similar cell activation pathway as NGF. To
investigate this possibility, we treated cells with a suboptimal dose ofNGF along with
increasing doses of CLN. The neurite outgrowth due to the combination of the NGF
and CLN was only slightly higher than that NGF or CLN alone, suggesting that there
is no synergism between these agonists. These data also suggest that the cell surface
receptor(s) may be different. NGF impacts cells through its high affinity receptor,
TrkA in PC12 cells (Klesse and Parada, 1999; Szeberenyi, 1996; Vaudry et al., 2002).
To test further whether CLN binds to TrkA, we inhibited cellular actions by K252A.
K252A attenuates activity of the Trk tyrosine kinase activated by NGF (Ohmichi
et al., 1992). Incubation of NGF-treated PC12 cells with 100 nM K252A caused a
rounded phenotype, and no neurite outgrowth was observed; however CLN-induced
neurite outgrowth was not significantly affected by K252A. These data suggest that
the cellular actions of CLN were not dependent on K252-sensitive pathways. It has
recently been shown that integrated signaling through extracellular signal-regulated
kinase (ERK), c-Jun N-terminal kinase (JNK), mitogen-activated protein kinase,
and additional Ras-dependent signaling pathways distinct from the ERKs and JNKs
may contribute to agonist-dependent gene expression in PC12 cells (Marek et al.,
2004; Riese et al., 2004; Xiao and Liu, 2003). Future studies are needed to define the
CLN receptor, whose activation leads to action of p53–p21WAF1 pathway, cell cycle
arrest, and downstream events characterized by neurite outgrowth in PC12 cells. It
is interesting to note that CLN alters oxidative stress-induced activation of the JNK
and MAPK pathways (Boldogh et al., 2003a).
In this study, we compared CLN’s activity in inducing neurite outgrowth toNGF
in cultures of PC12 cells. We found that 50 ng/mL CLN resulted in 30% of the cells
showing neurite extensions, while 90% of the cells differentiated after the addition
of identical dose of chemically uniform NGF. Proposing that the neurite outgrowth
by CLN was due to a single constituent, we assume that CLN’s active principle is
more effective than that of NGF. It is also possible that multiple peptides of CLN
are responsible for neurite outgrowth of PC12 cells. In the event of the validity
of the latter scenario, a synergistic or additive effect of these as-yet unidentified
peptides will be one of our challenging tasks for future research. Nonetheless, an
uncovering of the cell-differentiating activity of CLN derived from initial mother’s
milk (colostrum) is novel. The potential clinical applications of CLN make the PC12
cell-based neurite outgrowth assay important in identification of the active principle
and this assay attractive for determining uniformity of various CLN preparations,
along with two-dimensional polyacrylamide gel analysis.Uniform CLN preparations
in hand will mollify the concerns about complex nature of CLN and reproducibility
of their behaviors in cell culture models.
Finally, these studies show for the first time that CLN inhibits cell proliferation
and induces morphological changes in PC12 cells. The neurite outgrowth by CLN
appears to involve p53 and p21WAF1. We propose that CLN mediates a wide spectrum
of activities similar to those induced by hormones and neurotropins leading to
neurite outgrowth. Because the signaling pathways involved appear to be common
to regulation of cell proliferation and differentiation, it suggests the potential use
of CLN to modulate gene expression that may be required for the development,
maintenance, and regeneration of neurons in the central nervous system. Recent
publications have described that treatment with CLN, a chemically uniform mixture
of low-molecular weight polypeptides improved symptoms in Alzheimer’s patients
with mild-to-moderate dementia (Bilikiewicz and Gaus, 2004; Leszek et al., 1999).
Further work will be necessary to make correlations between the effects of CLN on
cell differentiation and maintenance/regeneration of neurons in vivo.
ACKNOWLEDGMENTS
This work was supported by ReGen Therapeutics, Plc, London, England and
the NIEHS Center at the University of Texas Medical Branch at Galveston, Texas
(Grant No. ES06676). We are grateful to Dr. David Konkel for scientific/editorial
advice and corrections made in the manuscript
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