Biomedical Engineering Reference
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activation of the SRC family kinase (SFK) and PI3K/AKT signaling pathways
(Braydich-Stolle et al.
2007
; Lee et al.
2007
; Oatley et al.
2007
). Pharmacological
impairment of SFK signaling blocks GDNF up-regulation of
Bcl6b
,
Etv5
, and
Lhx1
gene expression, without effecting cell survival or expression of non-GDNF-regu-
lated genes, indicating a specific role in SSC self-renewal (Oatley et al.
2007
). In
contrast, pharmacological impairment of PI3K/AKT signaling induces apoptosis in
cultured SSCs and impairs expression of both GDNF and non-GDNF-regulated
gene expression, indicating a general role of PI3K/AKT signaling in cell survival
rather than SSC self-renewal (Oatley et al.
2007
).
Recent studies by Lee et al. (
2009
) have identified RAS proto-oncogene as a
downstream signaling target of GDNF and FGF2 induced SFK stimulation in cul-
tured mouse gonocytes. Supplementation of culture medium with either GDNF or
FGF2 was equally effective at activating RAS. Additionally, gonocytes harboring
overexpression of an activated form of RAS are able to proliferate in the absence
of both GDNF and FGF2 stimulation and treatment with a pharmacological inhibitor
of MAPK signaling prevented RAS-induced cell growth. These findings are sur-
prising given that FGF2 effectively stimulates RAS activity but is unable to support
stem cell self-renewal in cultures of wild-type spermatogonia (Kubota et al.
2004b
;
Kanatsu-Shinohara et al.
2005
). Also, pharmacological inhibition of MAPK signaling
in cultures of wild-type gonocytes did not affect their growth, suggesting
in vitro
adaption of the RAS overexpressing cells that may not reflect self-renewal mecha-
nisms of stem cells
in vivo
(Lee et al.
2009
). Transplantation analyses of cultured
gonocyte populations overexpressing activated RAS revealed that stem cell content
was reduced by greater than 50% after 2 months of
in vitro
maintenance (Lee et al.
2009
). This finding suggests that RAS signaling alone is unable to completely
replace growth factor requirements for prolonged SSC self-renewal.
Stem cell proliferation in most tissue is an infrequent occurrence and self-renewal
cues may act by regulating progression of the cell cycle. Lee et al. (
2009
) found that
gonocytes overexpressing activated RAS had elevated levels of cyclin D2 expres-
sion. Similar to RAS overexpression, cyclin D2 overexpression in cultured gono-
cytes promoted growth factor independent proliferation, and transplantation analysis
revealed no decline in stem cell content after 2 months
in vitro
(Lee et al.
2009
).
These observations indicate that GDNF and FGF2 induction of SSC self-renewal
occurs, at least in part, through regulation of entry into G1 phase of the cell cycle.
Because RAS lacks a DNA binding domain, direct regulation of cyclin D2 transcrip-
tion must occur through other intermediaries that have yet to be defined.
7.5
Summary
In the mammalian germline, SSCs undergo both self-renewal and differentiation to
support continual spermatogenesis from puberty until old age in males. Self-renewal
of SSCs is dependent on extrinsic stimulation by the growth factor GDNF. Our
current understanding of internal molecular mechanisms regulating SSC self-renewal
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