This study was designed to investigate the role of GH on postnatal neurogenesis using a neurosphere culture system. The presence of ongoing neurogenesis in the postnatal mammalian brain, together with the prospect that the stem cell pools present in the adult brain can be directed towards repair of neural injuries has generated much interest for the last several years, see [19, 33] for review. Furthermore, since postnatal neurogenesis is regulated by a complex network of signaling molecules and extrinsic factors , understanding these mechanisms of control will help to find effective treatments directed towards increase neurogenesis in order to promote brain repair.
GH has been proposed to play a role on brain repair after injury . Although some of the beneficial effects of GH on brain repair may depend on their neuroprotective actions , there is also accumulating evidence indicating that neurogenesis is also stimulated by GH. Thus GH treatment has been demonstrated to promote neurogenesis in different brain areas [20, 23, 24, 35] either under unperturbed conditions [20, 35] or in response to brain damage [23, 24]. Interestingly, GH-induced neurogenesis is potentiated when GH treatment is combined with physical exercise [35, 36] or physical rehabilitation , thus suggesting that the hormone is not only cooperating with endogenous mechanisms regulating postnatal neurogenesis, but may be even essential to induce the neurogenic response . There is also evidence indicating that GH increases the frequency of both rodent- and human-derived neurospheres in vitro [37–41]; and the present study], thus demonstrating a direct effect of GH on neural precursor cells. Furthermore, in all these cases, GH actions are lost when GHR is absent or inactivated, thus indicating that GH actions are exerted via activation of GHR; although the possibility that some of GH actions on neural precursors may depend on the activation of other receptors cannot be ruled out .
Once established the importance of GH in the regulation of proliferation and survival of SGZ-derived neurospheres, we next investigated the molecular mechanism underlying these actions. The predominant GH signal transduction cascade comprises activation of the GHR dimer and phosphorylation of Janus kinase 2 (JAK2). Activated JAK2 then phosphorylates key tyrosine residues on the cytoplasmic domain of the GHR, permitting SH2 domain interactions by signal adaptor proteins that results in docking of signal transducer and activator of transcription 5 (STAT5) for activation by JAK2 [25, 26]. The triggering of this canonical signaling pathway has been recently demonstrated in neurosphere cells , and may be involved in some of the GH actions on these cells. However, phosphorylation of the cytoplasmic domain of the GHR also results in the activation of the Ras/MAPK kinase/ERK pathway; while activated JAK2 is also able to directly phosphorylate IRS-1/2, facilitating activation of the PI3K/Akt/mTOR pathway [25, 26].
Our findings demonstrate that at least two of those signaling pathways, the PI-3 K/Akt/mTOR and JNK module of the MAPK, are activated by GH in cultured neurospheres, thus illustrating the complexity of GHR signaling in these cells. Furthermore, by using selective inhibitors we have been able to demonstrate that, despite these signaling pathways may be important in mediating the GH effects on cell proliferation, they appear to exert a more essential role in the regulation of cell survival. There are several types of evidences supporting our hypothesis. First, GH was able to partially overcome the effect of ERK inhibition on cell survival, while its effect on cell proliferation was completely abrogated under these circumstances. The ability of GH to promote cell survival in the absence of ERK signaling indicates that GH is able to deliver a survival signal that is independent on Ras/ERK pathway activation. In contrast, Ras/ERK signaling is necessary for the GH effect on neurosphere proliferation indicating that, in keeping with previous reports [41, 42], GH is neither sufficient nor necessary for neurosphere proliferation.
Second, GH treatment also partially counteracted the negative effect of rapamycin on cell survival, but did not modify rapamycin-induced inhibition of cell proliferation. Interestingly, mTOR has been shown to be relevant for EGF-dependent proliferation of neural precursors , further supporting our hypothesis regarding a minor role of GH on the regulation of neurosphere proliferation. Furthermore, mTOR is present in two distinct complexes: mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) [30, 44]. Since only mTORC1 is inhibited by rapamycin, the possibility exists that GH actions on cell survival may depend, at least in part, on the rapamycin-insensitive mTORC2 complex, as occurs with insulin . Another possible mechanism, not excluding the former, is that GH may overcome the effect of rapamycin treatment via the stimulation of survival signals that do not depend on the PI-3 K/Akt/mTOR pathway. In this regard, it is interesting to indicate that the ability of GH to prevent apoptosis was preserved in the presence of PI-3 K inhibition, thus suggesting that Akt activation does not play a major anti-apoptotic role in these cells.
In contrast with these findings, GH effects on both cell proliferation and survival were completely inhibited when the phosphorylation of JNK was blocked. JNKs are members of the MAPK family that are activated by a variety of environmental stresses, inflammatory cytokines and, in some instances, by growth factors and GPCR (G protein-coupled receptor) agonists . Once activated, the JNK pathway regulates numerous cellular responses including cell proliferation and survival, together with different aspects of neural development [47–50]. Therefore, the fact that blockade of JNK signaling completely inhibited both the proliferation and survival of SVZ-derived neurospheres, indicates that this pathway plays a major role in the regulation of the cellular fate of these cells. Furthermore, the fact that both GH-induced proliferation and survival are also blocked under these experimental conditions, together with the ability of GH to promote JNK phosphorylation, indicates that the JNK pathway is a key component of the signaling machinery activated by GH in SVZ-derived neurospheres. To our knowledge, this is the first time to demonstrate the capacity of GH to induce the phosphorylation of JNK in neural cells. To the present, GH-induced JNK phosphorylation has been demonstrated in macrophages , in NIH 3 T3 fibroblasts , and also in chinese hamster ovary cells stably transfected with rat GH receptor cDNA . However, GH has been shown to reduce both basal and doxorubicin-stimulated JNK transcriptional activity and phosphorylation in both MDA-MB-231 and MCF7 cells , thus demonstrating the complexity of GH-mediated JNK regulation.
Finally, it is noteworthy to indicate that blockade of GH signaling was able to induce a decrease in neurosphere proliferation and survival even in cells not receiving GH treatment. This finding, together with the existence of GH expression in cultured neurospheres (; and the present report), suggests the existence of an endogenous GH axis regulating the proliferation and apoptosis of neural progenitors. Up to now, the existence of GH immunoreactivity has been reported in several brain areas that include germinal regions of the embryonic brain , as well as brain regions involved in postnatal neurogenesis [54–56]. Furthermore, GH gene expression within these areas is increased by factors known to increase neurogenesis such as learning, exercise or estrogen administration (37; 54, 55), while such increase is not observed in GHR-/- animals (37). Altogether, these findings lead to the possibility that GH treatment is, in fact, cooperating with locally-produced GH in increasing the proliferation of neural progenitors in response to adequate stimuli. In this regard, it is interesting to note that we have previously found that GH is expressed in rat hippocampal progenitors, and that GH expression increases after neurotoxic damage (23). Therefore, it is tempting to speculate that, this locally-produced GH may cooperate with the exogenous hormone in promoting neurogenesis and cell survival in response to brain injury.