Immunophilins (IMMs) are a family of proteins that bind immunosuppressive drugs and possess cis/trans-peptidyl-prolyl-isomerase activity. They are classified as FKBPs (FK506-binding protein) when they bind FK506, and cyclophilins when they bind cyclosporine A (CsA) (Pratt et al. 2004a). FKBP51 and FKBP52 (gene names FKBP5 and FKBP4, respectively) are highly homologous tetratricopeptide-domain immunophilins whose sequences share 60% identity and 75% similarity. The best characterized interactions of FKBP52 and FKBP51 are with hsp90 and steroid receptor complexes. It has been shown that FKBP52 is required for dynein/dynactin-dependent retrograde movement of glucocorticoid receptor (GR) (Galigniana et al. 2004b), mineralocorticoid receptor (Piwien Pilipuk et al. 2007), p53 (Galigniana et al. 2004a), receptor-associated coactivator 3 (RAC3) and apoptosis-inducing factor (AIF) (Colo et al. 2008), and adeno-associated virus 2 (Zhao et al. 2006). Moreover, FKBP52 was found associated to androgen receptor (Cheung-Flynn et al. 2005), progesterone receptor (Kosano et al. 1998), estrogen receptor (Ratajczak and Carrello 1996), epithelial calcium channels (Gkika et al. 2006), and transient receptor potential (TRPC) channels (Sinkins et al. 2004). On the other hand, FKBP51 does not bind dynein (Wochnik et al. 2005) and shows inhibitory action of steroid receptor function, an effect that is abrogated by mutations of the tetratricopeptide domain. This suggests that the association with hsp90 is relevant for that biological property.
Immunophilins are also present in the brain at higher concentrations than in immune cells (Snyder et al. 1998). The Bruce Gold laboratory found that the IMM-binding drug FK506 (Tacrolimus) shows neurotrophic effect (Gold et al. 1999) and it was initially thought that this effect involved calcineurin inhibition. However, the persistence of the capability to hasten nerve outgrowth by FK506-derivatives devoid of immunosuppressive effects indicated that both effects are independent (Gold and Villafranca 2003). Experiments with mice where the FKBP1A gene was knocked-out clearly showed that such neurotrophic effect is not mediated by FKBP12, whereas the use of a monoclonal antibody against FKBP52 completely blocked the effect of FK506 (Gold et al. 1999). Even though this evidence suggests that FKBP52 could play a cardinal role in neuritogenesis, the molecular mechanism involving IMMs in neuronal differentiation and neurite outgrowth still remains totally unknown and there are no systematic studies to elucidate the cellular events related to this conundrum. In this study, we analyzed the subcellular redistribution of the FKBP52•hsp90•p23 heterocomplex in undifferentiated N2a neuroblastoma cells and rat embryonic hippocampal neurons stimulated with FK506, and reveal for the first time some of the subcellular events involving these chaperones during the differentiation process of neurons.
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Since the discovery that the immunosuppressant drug FK506 accelerates functional recovery and axonal regeneration in the rat sciatic nerve-crush model, the mechanism related to cell differentiation and neuritogenesis has remained enigmatic and not fully addressed to date. N2a neuroblastoma cell line and embryonic hippocampal E17 neurons provide a superb model to study molecular programs along differentiation. Here, we show for the first time a very peculiar property of undifferentiated neuronal cells. The FKBP52•hsp90•p23 complex forms a perinuclear ring that undergoes a rapid subcellular redistribution along the cytoplasm, FKBP52 being concentrated in terminal axons and arborization areas. In agreement with the potential importance of this IMM inferred from its subcellular redistribution, knock-down experiments showed that FKBP52 play a key role in the architecture of these nuclear rings as these structures faded in most cells (if not all of them) where the expression of this IMM was abrogated (Fig. 11d). Importantly, both the rate of cell differentiation and neurite outgrowth were also inhibited.
There is a direct relationship between the disassembly of the chaperone complex and the progression of the differentiation, the chaperones migrated to the cytoplasm being associated with cytoskeletal rearrangement, whereas the nuclear areas occupied by them in undifferentiated cells become transcriptionally active. In contrast to FKBP52, FKBP51 is not induced during differentiation, remains in the cell body and replaces FKBP52 in the annular structures of the nucleus complexed with hsp70. Importantly, these effects are triggered by FK506 itself without need of other trophic factor.
Initially, it was assumed that the mechanism underlying neurite outgrowth by FK506 was via calcineurin inhibition. However, there were developed a number of ligands lacking immunosuppressant activity that retain the neuroprotective activity (Gold et al. 2005; Voda et al. 2005; Price et al. 2007). It was proposed that the effect of FK506 is mediated by FKBP52 and not by other FKBPs (Gold 1999; Manabe et al. 2002; Price et al. 2005; Kang et al. 2008). Our observations are in line with this hypothesis and also add the fact that FKBP51 seems to antagonize FKBP52 action. It has also been posited that FK506 only works in the presence of neurotrophins. Our experimental evidence shows that FK506 does exert neurotrophic action by itself in both neuroblastoma cells and embryonic hippocampal cells, an effect that seems to be associated with the re-localization of the IMMs and the generation of new protein-protein interactions, some of them essential for the rearrangement of the cytoskeletal architecture. Good examples are the association of p23 with intermediate filaments and the disruption of microtubules and microfilaments in arborization bodies (Fig. 9).
Previous works have related the activation of the MAPK pathway with the mechanism of action of FK506 (Matsuda and Koyasu 2003; Price et al. 2003; Gold and Zhong 2004), although the effect was always mixed with that dependent on neurotrophins. Most of the observations made in our study for FK506-induced differentiation parallel those observed in cells stimulated with cAMP. However, it is clear that the events related to cell differentiation do not share the same mechanism.
The ERK cascade is activated during the early steps of differentiation and this activation is linked to the redistribution of chaperones from the nucleus to the cytoplasm (Fig. 6a and b). Even though the biochemistry of ERK pathways is well known, how the activation of this pathway achieves different specific responses is unclear. This concept is not only valid for neuronal differentiation, but also for most signaling pathways related to ERK cascade.
The over-expression of FKBP52 induced faster cell differentiation and neurites were longer. The opposite action was observed after knocking-down FKBP52. On the other hand, FKBP51 over-expression decreased both the length of neurites and the rate of cell differentiation, and its knock-down favored neurite outgrowth. FKBP52 (and not FKBP51) concentrates in the growing cones, neurite varicosities that will originate arborization, and the terminal end of axons of neurons or axon-like structures of the cell line.
FK506-binding proteins are highly abundant in the nervous system (Snyder et al. 1998). One of the roles recently reported for FKBPs is to interact with proteins belonging to the TRPC superfamily. The association of FKBP12 and FKBP52 with TRPC channels in rat brain lysates is displaced by FK506 (Sinkins et al. 2004). As FK506 disrupts both the binding of the IMM to TRPC channels, and it also inhibits the peptidyl-prolyl isomerase (PPIase) activity and calcineurin activity, it is unlikely that such inhibitory action of FK506 may be responsible for neuron differentiation, neurite outgrowth and nerve regeneration because all these effects are also seen in FKBP12-KO mice or with FK506 derivatives lacking the ability to activate calcineurin. Our results suggest a more complex network of biological processes in addition to the possible (and tempting) speculation that activity of FKBP52 action may be related to some gating mechanism of those channels. TRPC channels are not exclusive of neurons, but they are widely expressed in several cell types (Parekh and Putney 2005), whereas the effects of FK506 on cell differentiation via hsp90-binding immunophilins are not observed in other tissues and seem to be exclusive for neurons. A good example is shown in Fig. 3(e) with L1 pre-adipocytes, a cell line that expresses TRPC channels. It is important to emphasize that the phenomenon described in this study involves an entire rearrangement of neuronal chaperones that may affect simultaneously several aspects of the differentiation physiology, that is, from gene regulation (Fig. 7) to cytoskeleton organization and axonal growth (Figs 8, 9 and 11). In this regard, it is likely to postulate that these chaperones may repress gene expression in the annular structures observed in the nucleus of undifferentiated neurons, for example, by favoring the condensation of chromatin. In the cytoplasm, the chaperones acquire a different role influencing the organization of the cytoskeleton, in particular in dendrite bifurcations and terminal axons.
A very recent report has suggested a potential role of IMMs in axon guidance in response to netrin-1 (Shim et al. 2009). Both processes, axon guidance via netrin-1 and the particular rearrangement of the FKBP52•hsp90•p23 complex during the early neuronal differentiation steps, are not directly related as it has also been shown that axons continue to grow after the over-expression of FKBP mutants that blocked the responses to netrin-1, demonstrating that axonal growth was not impaired. Moreover, netrin signaling requires the activity of soluble adenylyl cyclase (Wu et al. 2006), whereas FK506 seems to be independent of this pathway. Rather, our study shows univocally that axonal growth is abrogated according to the balance between FKBP51 and FKBP52, key factors for neuronal differentiation as well.
Recent studies indicates that the periphery of the nucleus provides a platform for sequestering transcription factors away from chromatin when these factors interact physically with components of the inner membrane (Heessen and Fornerod 2007). Additionally, it has been proposed that the nuclear periphery is an epigenetically dynamic compartment containing both active and repressed genes (Luo et al. 2009). The lamins are developmentally regulated, with all cells expressing B-type lamin, whereas A-type lamins are absent in early embryonic development and in certain stem cell populations in adults (Stewart et al. 2007). This is why we performed the studies of co-localization labeling lamin B. Our findings suggest that the FKBP52•hsp90•p23 complex may shape the nuclear architecture of undifferentiated cells, such that the initiation of the differentiation program promotes the redistribution of the components of the complex, thus affecting gene expression. In line with this hypothesis, pre-mRNA labeling show high and concentrated transcriptional activity in the perinuclear area where the chaperone complex is disassembled before being dispersed in the cytoplasm.
The hsp90-inhibitor radicicol shows some initial effect on neuronal differentiation, but it results toxic in the long term and also impaired FK506 effects (Fig. 1). Previous studies have used hsp90-inhibitors as differentiation agents for neurons, but the stimulation system was a mixture of drug and neurotrophic factors (Gold et al. 1999; Sano et al. 1999). Another important difference was that these studies were performed within a time-frame when inhibitors do not show the deleterious action described in Fig. 1 (Jin and Sano 2008). Because the MEK-ERK-CREB pathway seems to be involved in the FK506-dependent differentiation process (Figs 1 and 6), one possible candidate to initiate the activation of this cascade is Ras/Raf (Kolch 2000; Pursiheimo et al. 2002; Price et al. 2003). However, the activation of these kinase pathway is entirely dependent on hsp90 (Pratt 1998), so experiments with hsp90 inhibitors such as radicicol or geldanamycin should have prevented neurite outgrowth, which is not the case shown neither here nor by other laboratories. Consequently, FK506 must act down-stream of this pathway.
It has been proposed that the effect of hsp90-inhibitors on the neurite outgrowth may be related to the disruption of steroid receptors (Gold and Villafranca 2003), such that the released chaperone heterocomplex may work via a gain-of-function mechanism. Thus, it was proposed that upon dissociation from the receptor complex, the chaperones may favor retrotransport of signaling endosomes via FKBP52-dynein (Gold 2002) in a similar manner as it has been shown for steroid receptors (Galigniana et al. 2010). However, it should be noted that chaperones are not limiting proteins; actually, they are the most abundant soluble proteins and are greatly induced during the differentiation of neurons (Fig. 1e). Moreover, cells differentiate in the same extend with or without dexamethasone (Fig. 2c and d). Even though the influence of non-genomic effects of steroid hormones cannot be ruled out in the long term, the observation that the FKBP52•hsp90•p23 heterocomplex undergoes a dramatic subcellular rearrangement makes more likely that they (FKBP52 in particular) have specific functions on the early steps of the differentiation process. This interpretation is enhanced by the antagonistic action shown by FKBP51, an IMM that shares many structural properties with FKBP52. Therefore, we propose that IMMs are involved in the very early steps of activation of key genes specifically activated during neuronal differentiation and also participate in the rearrangement of the cytoskeleton.
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Figure S1. (a) N2a cells were stimulated with 1 μM FK506 for 3 h or 24 h. The right panel (Aged) corresponds to incubations performed with a medium reused in a previous incubation of 72 h. (b) Toxic effects of cyclosporine A (CsA) or radicicol (RAD) can also be seen after 24 h of incubation with concentrations five times lower than those used in Fig. 1(a). Cells treated with 1 μM FK506 for the same period of time are shown for comparative purposes.
Figure S2. Bar graph for neurite lengths measured in identical conditions as those described for Fig. 2(a) except that the incubation time was 24 h.
Figure S3. Subcellular distribution chaperones in 3T3-L1 pre-adipocytes is not affected by 4-h treatment with a differentiation cocktail. Bar = 10 μm.
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