Insulin-like growth factor-1 (IGF-1), also called somatomedin C, is one of the most important stimulators of cell growth (Yakovchenko et al. 1996), and a potent inhibitor of programmed cell death in both neuronal and non-neuronal cells (Morales et al. 2000; Tu et al. 2010). Although liver is the primary source of circulating IGF-1, significant expression of this growth factor is observed in various organs such as brain where it is known to exhibit autocrine and paracrine functions (D'Ercole et al. 1996). IGF-1 exerts its cellular effects by binding to its type I receptor, which subsequently activates two main downstream signaling pathways, namely the RAF-MEK-extracellular signal-regulated kinase phosphorylation cascade and the phosphatidylinositol 3-kinase-AKT pathway (Zheng et al. 2000; Leinninger and Feldman 2005).
The cAMP response element binding protein (CREB) is one of the common nuclear targets of the extracellular signal-regulated kinase pathway and the PI 3-kinase signaling pathway (Johannessen et al. 2004). It is a 43-kDa nuclear transcription factor belonging to the CREB/ATF family that regulates the transcription of the downstream genes (Kulik et al. 1997). There is a large amount of evidence to suggest that CREB plays a significant role in memory formation, stabilization, and retention (Yin et al. 1994; Bartsch et al. 1995). CREB controls the transcriptional responses of neurons to many extracellular stimuli, such as IGF-1 (Balschun et al. 2003) and also participates in synaptic plasticity and memory consolidation (Mozzachiodi and Byrne 2010). Hippocampal CREB gene transfer increases CREB expression and significantly improves the learning and memory function in aging rats (Mouravlev et al. 2006). PC12 cell line is a commonly used model of neuron derived from a transplantable rat adrenal phaeochromocytoma. It has been showed that IGF-1 mediated phosphorylation and transcriptional activation of CREB in PC12 cells and IGF-1 leading to CREB1-dependent neuronal specific gene expression (Pugazhenthi et al. 1999).
The microRNAs (miRNAs) are a group of small non-coding RNAs that are single stranded chains consisting of 19–25 nucleotides (~ 22 nucleotides) and transcribed by RNA polymerase II or III in the nucleus (Lee et al. 2004). More and more studies showed that miRNAs play an important role in gene regulations by binding imperfectly to the 3′-untranslated region (3′-UTR) of the target mRNAs, which leads to either translational repression or target mRNA cleavage (Lee et al. 1993).
microRNA-181 family, including miR-181a, miR-181b, miR-181c, and miR-181d, is one of the identified miRNAs that widely exists in vertebrate cells. miR-181a was found highly expressed in adult mouse brain tissues (Miska et al. 2004). miR-181a, showing different expression profiles at different stages of human neurodevelopment, was highly expressed in mature human neurons (Smith et al. 2010). These studies strongly suggest that miR-181a is closely related to brain cell development and differentiation. The bioinformatics analysis showed that CREB1 mRNA 3′UTR contains complementary sequence to the miR-181a seed region. In addition, IGF-1 regulated the expression of CREB to delay the arrival of cognitive and memory dysfunction with the aging (Anderson et al. 2002).
To understand whether miR-181a is a negative regulator for CREB1 expression in neurons, the expression of IGF-1, CREB1, and mature miR-181a in the hippocampus of Lewis rat were analyzed in this study, with the effect of miR-181a on dendrites growth of neurons and the mechanism of miR-181a in IGF-1 induced activation of CREB1 in PC12 cells investigated.
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IGF-1, as a potent neurotrophic factor, plays a critical role in the development and maturation of the CNS by promoting survival and proliferation of many types of brain cells (Guan et al. 2003). Transgenic and gene knock-out mice study indicates that IGF-1 plays a key role in the CNS development and helps to reduce the risks of gliomas and neurodegenerative diseases (Russo et al. 2005).
miRNA is involved in regulation of the CNS development in a temporal and spatial order; and at the same time, it is also participates in maintenance of various different nerve cells in the normal morphology and functions. miRNA expression has conservative, short-term and tissue-specific features in CNS (Kapsimali et al. 2007). miR-181a was highly expressed in mature neurons and adult mouse brain (Miska et al. 2004; Smith et al. 2010). CREB is the converged point of many signal pathways in hippocampal neurons and plays a crucial role in learning and memory (Disterhoft and Oh 2006; Mozzachiodi and Byrne 2010). Bioinformatics analysis showed that there is a possible target of miR-181a in CREB1 mRNA 3′UTR sequence. At present, the exact function of miRNA-181a in hippocampus has not been cleared and its role in regulation of CREB1 remains to be determined. This study confirmed that CREB1 mRNA 3′UTR is a direct target of miR-181a, suggesting a role of miR-181a in regulation of CREB1 expression.
Previous studies have suggested that in hippocampus or brain, IGF-1 protein expression is maybe growth hormone-independent and its autocrine is an important regulatory factor in hippocampal neurogenesis (Sun 2006). In the present study, the expression of miR-181a, IGF-1, and CREB1 in hippocampus of newborn and adult Lewis rats were examined. The results revealed that IGF-1 protein levels were significantly higher in adult Lewis rats compared with the newborns, and the total and phosphorylated CREB1 were approximately tripled in the hippocampus of adult rats. While miRNA microarray and quantitative RT-PCR showed that miR-181a expression was obviously lower in adult Lewis rats in comparison to the newborns. These findings suggested that an enhanced expression of CREB1t by higher levels of IGF-1 in hippocampus of adult Lewis rats is related to an inhibited miR-181a expression.
To further confirm the relationship between the inhibited miR-181a and up-regulated CREB1, this study then investigated whether miR-181a can bind and affect the predicted target CREB1 mRNA through interactions with its 3′UTR. Accordingly the mRNA 3′UTR of CREB1 was cloned into a reporter vector, downstream of a firefly luciferase cDNA. It was demonstrated that in HEK293 cells, the vector pGL3C-CREB1 3′UTR containing the 3′UTR of CREB1 mRNA displayed a clear reduction of light emission upon co-transfection with miR-181a mimic. On the other hand, co-transfection of scramble miRNA or miR-181a mimic with pGL3C-CREB1 3′UTR mutant did not induce obvious changes in the luciferase activity. These indicate the presence of a direct binding site for miR-181a.
As demonstrated by western blot analysis, when miR-181a mimic or miR-181a inhibitor was transfected into PC12 cells, CREB1 was inhibited by over-expressed miR-181a or enhanced upon miR-181a repression. In primary cultured hippocampal neurons, dendritic growth was obviously induced by transfection of miR-181a inhibitor and blocked, by miR-181a mimic. These findings, together with the previous evidence that dendritic growth is regulated by activation of a transcriptional program that transduces CREB1-mediated signaling into the nucleus (Redmond et al. 2002), support a negative effect of miR-181a on dendritic growth of neurons by regulating CREB1 gene.
From worms to humans, IGF-1 has important roles in CNS development. It promotes the proliferation, survival, and differentiation of all types of brain cells (D'Ercole et al. 1996). Pugazhenthi et al. (1999) reported that IGF-1 mediated transcriptional activation of CREB1 within a time less than 30 min, in rat PC12 cells. Here, this study demonstrated that IGF-1 up-regulated CREB1 protein expression and phosphorylation at 24 h in the same cell line with a maximal effect at a concentration of 50 ng/mL. In addition, this study also demonstrated that IGF-1 had a negative effect on miR-181a expression. These data, together with those mentioned above, suggest that exogenous IGF-1 can markedly up-regulate CREB1, at least partially, by inhibiting miR-181a expression. There are numerous studies demonstrated that other miRNAs participated in regulating CREB1 protein expression (Leone et al. 2011; Kong et al. 2012). And CREB1 plays a key role in learning and memory. All these add a complex non-coding RNA regulation of CREB1 gene to those by traditional cytokines.
IGF-1 and CREB1 activities are critically reduced in the context of aging and of age associated brain diseases (Caccamo et al. 2010; Piriz et al. 2011). As a potent neuroprotective agent, IGF-1 promotes neuronal cell health by triggering genetic programs that are largely dependent on CREB1 (Carro and Torres-Aleman 2004). This research shows that IGF-1 can enhance CREB1 expression but inhibit the generation of mature miR-181a. And in contrast, miR-181a has a negative effect on CREB1 expression, which indicates an important role for miR-181a in neurodegeneration. This find may direct to a new therapeutic target for the prevention of age-associated cognitive impairment and neurodegeneration.
In summary, this study demonstrated for the first time that miR-181a is involved in IGF-1 mediated regulation of CREB1 expression through an interaction with CREB1 mRNA's 3′UTR; and its mimic inhibits dendritic growth of hippocampal neurons. These findings suggest miR-181a as a potential target for preventing neurodegenerative diseases.