Surprisingly, GH cotreatment reduced the expression of the proliferation marker PCNA in this short-term protective protocol only when excitotoxic damage was induced; however, contrary to expected, cell proliferation did not increase due KA injury alone. On the other hand, we observed an upregulation of FGF2 gene expression after KA injection. This response is in accord with previously reported effects of NDMA on FGF2 expression.
33,49,50 When GH was coinjected with KA, FGF2 mRNA levels were significantly reduced and returned to levels similar to that of the control group, indicating that GH negatively regulates its expression. We subsequently confirmed this finding in QNR/D cells overexpressing GH and in embryonic primary retinal cell cultures treated with GH. This finding is important as it has been reported that FGF is a potent regulator of Müller cell transdifferentiation and proliferation, with multiple injections of FGF2 being able to cause MGPC formation even in the absence of neurotoxic insult.
27,29,33,34,50,51 FGF2 acts mainly on the Müller glia by upregulating the complex network of signaling pathways, which promote transdifferentiation, including Notch, MAPK, pCREB, and β-catenin.
19–21,52 In addition, we found that the expression of Sox2 in our model mirrored the expression of FGF2. Sox2 is a transient transdifferentiation marker only expressed by Müller cells in response to damage and/or repeated doses of growth factors.
25,33,53 We have previously proposed a model of GH action on the retina, where it regulates the local expression of endogenous growth factors, which then mediate its neuroprotective activity in this tissue, in addition to its direct effects on RGCs.
8,12 Our current results strengthen and support this model, showing that the negative regulation of FGF2 expression by GH leads to the inhibition of MGPC formation and proliferation. It should be noted that GH has been shown to exert well-established actions promoting regeneration and proliferation of progenitor cells in other models.
54–59 Indeed, it is known that GH is upregulated in proliferating rat hippocampal progenitor cells after KA damage, with the proliferative response further enhanced by GH treatment.
55 The observation that GH blocked a classical growth factor, such as FGF2, and exerted antiproliferative effects was an unexpected finding; thus, the analysis of other timeframes, treatment protocols, identification of secreted factors, and other intracellular signaling pathways is mandatory to understand this multifactorial neurotrophic effect. The downregulation of FGF2, Sox2, and Ascl1a, together with the upregulation of BMP4, suggests a neuroprotective effect that blocks or delays the proliferative response in order to generate neural progenitors.
33,60