In a previous study, we have used PC12 cells to examine the effects of IL-6/s-IL6R on neuronal differentiation in comparison to NGF [22
]. Already after 24 hours of exposure to IL-6/s-IL-6R or NGF PC12 cells are highly active in cellular growth and proliferation displaying pronounced formation of extending neurites. Combined incubation with IL-6/s-IL-6 plus NGF drastically enhanced cell number and neurite outgrowth arguing for an additive effect of both stimuli on neuronal differentiation. In the current study we have chosen this time point to perform microarray analyses in order to monitor changes in gene expression and to compare the genetic programs utilized for neuronal differentiation by IL-6 versus NGF.
An important aspect in gene expression profiling using microarrays is the accuracy of the measurements in the relative changes in mRNA expression. Thus, alternative technologies such as qRT-PCR are used for the validation of microarray data [27
]. Several systematic studies comparing the changes in gene expression obtained from oligonucleotide- or cDNA arrays to data from qRT-PCR revealed that a good correlation exists for genes exhibiting fold-change differences in expression of > 2-fold [28
]. Therefore, in our datasets all genes displaying changes in expression levels of < 2-fold were excluded. Moreover, our exemplary validation data on GAP-43- and REG3B-expression are in line with other previous reports confirming that it is rather the magnitude of fold change varying between qRT-PCR and Affymetrix-analysis, but not the direction.
Detailed Ingenuity biological function analyses reveal that IL-6 and NGF activate gene sets that regulate the same process in neuronal differentiation and nervous system development, however, utilizing completely distinguished sets of individual molecules. This may explain our previous observation that combined application of IL-6/s-IL-6R plus NGF generates an additive effect on PC12 cell differentiation. Important processes in neuronal differentiation and nervous tissue development include cellular growth and proliferation in order to enhance cell number. Neurite outgrowth and network generation requires migration of neurons or nerve growth cones. Neuronal navigation is guided by the interaction of the neuron with its local environment, in particular by chemotaxis as the key mechanism. This process involves three major steps including directional sensing along a gradient of chemotactic factors, cellular motility i.e. the cell's movement by changes in cytoskleletal organisation and cellular adhesion and cellular polarisation [30
]. Certainly, a key step in the regulation of these processes is the increased gene expression of growth factors and functionally related external molecules, indicating convergence of several different signaling pathways (Table ). In IL-6 stimulated PC12 cells these tasks may be taken by growth differentiation factor 15 (GDF15), platelet-derived growth factor alpha (PDGFA), hepatocyte growth factor (HGF), regenerating islet-derived 3 alpha (REG3A), regenerating islet-derived 3 beta/pancreatitis-associated protein I (REG3B/PAPI), growth hormone releasing hormone (GHRH) and adenylate cyclase activating polypeptide (PACAP). NGF recruits GDF15 (131-fold), transforming growth factor beta 1 (TGFB1; 101-fold) and brain-derived neurotrophic factor (BDNF; 89-fold). TGFB1 is the prototype member of the TGFB-superfamily comprising multifunctional growth factors with numerous cell and tissue functions such as cell cycle control, regulation of early development, differentiation, extracellular matrix (ECM) formation and chemotaxis. In the nervous system, TGFB1 has been shown to regulate neuroprotection against glutamate cytotoxicity, ECM production, and cell migration in the cerebral cortex, control of neuronal death as well as survival of neurons (reviewed in [33
]). GDF15 is a member of the TGFB- superfamily and has been shown to be a potent trophic factor in the brain (reviewed in [34
]). Hepatocyte growth factor (HGF) is a chemoattractant and a survival factor for embryonic motor neurons. In addition, sensory and sympathetic neurons and their precursors respond to HGF with increased differentiation, survival and axonal outgrowth [35
]. Moreover, HGF may synergize with other neurotrophic factors to potentiate the response of developing neurons to specific signals [36
]. Platelet derived growth factor (PDGF) has been suggested to support neuronal differentiation [37
], and has previously been reported to act as a mitogen for immature neurons [38
] and neural progenitor cells [39
]. REG3A and REG3B/PAPI are members of the regenerating protein (REG)/pancreatitis-associated protein (PAP) family representing a complex group of small secretory proteins which display many different functions, among them growth factor activity for neural cells [40
]. So far, only limited knowledge is available about the role and function of PAP/REG-proteins in the nervous system. REG3B/PAPI expression is induced in spinal motor neurons as well as subsets of the dorsal root ganglion neurons [41
]. Moreover, in vitro
REG3B/PAPI has a mitogenic effect on Schwann cells [42
]. In a hypoglossal nerve injury model in rats, expression of REG3B/PAPI mRNA was found to be enhanced in injured motor neurons after axotomy and a marked induction of REG3G/PAPIII mRNA was observed in the distal part of the injured nerve [43
]. More recently, REG3G/PAPIII has been identified as a macrophage chemoattractant that is induced in and released from injured nerves [44
]. With REG1A/PSP and REG3G/PAPIII, two further members of the REG/PAP family are induced by IL-6 in PC12 cells. It is noteworthy that these genes are up-regulated at the highest levels obtained in the entire dataset for IL-6. In NGF-treated PC12 cells, no up-regulation of the PAP/REG protein genes was observed. The results in our study are in line with an earlier report demonstrating up-regulation of PAP/REG gene family members in PC12 cells upon stimulation with IL-6/s-IL-6R [45
So far various studies have investigated gene expression profiles in NGF-treated PC12 cells applying different experimental protocols in respect to time points and periods of NGF administration [46
]. From most studies, it is obvious that PC12 cells require at least 3 to 5 days of NGF-treatment to obtain the fully differentiated neuronal phenotype. The most significant morphological changes occur within the first 2 days, reaching a plateau phase at day 3 [51
]. Redundant data sets as well as unique genes have been identified and followed. Our study provides novel candidate genes activated in the early phase of the differentiation process and thus may enlarge the repertoire of known NGF-regulated genes.
The current study reveals novel aspects of IL-6 action, notably that it applies several major routes to direct PC12 cell differentiation. Besides up-regulation of growth factors known to act in autocrine and paracrine fashion to take over further tasks in the differentiation process, these include induction of PACAP, a pleiotropic molecule with a broad spectrum of biological functions. Among them are actions as a neurotrophic factor similar to NGF as well as induction of transcription factors known to be of key importance in neuronal differentiation [52
Upregulation of PACAP could have an important impact on IL-6-induced PC12 cell differentiation. A recent report provided data from microarray analyses of PACAP-regulated gene transcripts in primary cultures of sympathetic neurons at 6 hours and 92 hours of stimulation [53
]. A comparison with our data reveals that many gene families that are activated by PACAP in primary sympathetic neurons are also induced by IL-6 in PC12 cells (Table ). Thus, many of the effects of IL-6 on PC12 cells are likely to be mediated by the intermediate autocrine and/or paracrine action of PACAP. PACAP is a member of a family of neuropetides known to activate class II G-protein coupled receptors (GPCRs; reviewed in [54
]). Other family members include growth hormone releasing hormone (GHRH) and calcitonin-related peptide beta (CALCB) which are activated by IL-6 in PC12 cells by 31-and 195-fold, respectively. All members of the class II GPCR superfamily regulate intracellular cAMP-levels by receptor coupling to the Gs-adenylate cyclase-cAMP signaling pathway [54
]. A further mechanism of PACAP action in PC12 cells could be a transactivation of TrkA receptors [55
]. However, in light that the overlap in the datasets of IL-6 versus NGF is rather small, TrkA activation may not be a primary event at all or at the time point of our study.
Table 6 Comparison of commonly regulated gene families in PACAP-stimulated sympathetic primary neurons versus IL-induced PC12 cells (data derived from )
A further key step in IL-6 actions on PC12 cell differentiation is the induction of RARA and EGR-1/Zif268, two transcription factors known to be of crucial importance in neuronal differentiation. Among the genes regulated by retinoic acid is GAP-43, a neuron specific protein frequently used as a marker of neuronal differentiation as it is expressed in most neurons during neuronal development, nerve regeneration and LTP [56
]. The data herein are confirmative to our previous study in which we have found induction of GAP-43 mRNA upon stimulation of PC12 cells with IL-6/s-IL-6R [22
]. EGR-1/Zif268 is induced in nearly every model of long-lasting synaptic plasticity in the CNS [61
] and suppression of Zif268 prevents neurite outgrowth in PC12 cells [65
]. Recently candidate target genes of Zif268 in PC12 cells were identified suggesting that a key component of the long-lasting effects of Zif268 on CNS plasticity is the regulation of proteasome activity [66
Signal transducer and activator of transcription 1/2 (STAT1/2), two members of the STAT family of transcriptions factors involved in signaling by Interferons (IFN) [68
] are activated by stimulation of the PC12 cells with IL-6. As we could not detect changes in IFN gene expression, an autocrine action of PDGF is the most likely candidate for upregulation of STAT1/2 as described for neural progenitor cells [39
]. STAT1/2 may upregulate interferon regulatory factor 1(IRF1)-expression, a further transcription factor of IFN-signaling. Breast cancer 1 (BRCA1) encodes a tumour suppressor gene whose germ line mutations in women are associated with a genetic predisposition to breast and ovarian cancer. STAT1 transcriptional activity is decreased by a physical interaction with BRCA1 as a key step in the regulation of IFN-induced cellular growth arrest [69
]. By the action of IL-6, BRCA1 gene expression is down-regulated thus supporting STAT1 mediated PC12 cell growth. We failed to detect STAT3 expression, the key transcription factor of IL-6 signaling. This is most likely due to the fact that STAT3 gene transcription occurs very early in IL-6-stimulation and is already terminated at the time point of the analysis, or the expression levels are below 2-fold and thus did not meet the exclusion criteria.
The morphological changes during nervous system development are controlled by interactions of individual neurons with the ECM. Signals from the ECM into a particular neuron are mediated by integrins via associated adapter molecules. In this way growth factor induced receptor tyrosine kinase (RTK)- and integrin-mediated signalling determine the fate of a particular cell, notably differentiation, cell shape, adhesion, polarity, migration, as well as proliferation versus apoptotic cell death (reviewed in [70
]). LIM and senescent cell antigen-like domains1/PINCH (LIMS1/PINCH) is an intracellular adaptor molecule providing the molecular link of an integrin-RTK network. LIMS1 physically connects integrin-linked kinase (ILK) to non-catalytic (region of) tyrosine kinase adaptor protein 2 (Nck2), an adapter molecule of the growth factor receptor (RTK) [70
]. LIMS1 is activated by IL-6 as well as NGF and thus is one of few genes regulated in the common subset. In contrast to IL-6, NGF simultaneously up-regulates major components of the ECM including collagen, type XI, alpha1 (COL11A1), COL12A1, fibronectin1 (FN1) as well as fibrillin2 (FN2) (Table ).
In contrast to NGF, only one publication provided expression profiling data analysing gene sets regulated by IL-6 upon neuronal differentiation. Primary cultures of rat dorsal root ganglia (DRG) were treated with IL6RIL6 for 2 and 4 days, respectively. A detailed comparison reveals that only a small number of commonly regulated genes may be identified in the datasets that are regulated in parallel or opposite direction. These include Egr-1 (upregulated in PC12 cells; downregulated in DRG cells), TGFA (upregulated in PC12 cells and DRG cells), TGFB (upregulated in PC12 cells; downregulated in DRG cells), PDGFA (upregulated in PC12 cells; downregulated in DRG cells) and IRF-1 (upregulated in PC12 cells and in DRG cells) [24
The results obtained from our study may also have impact into clinical treatments of injured peripheral nerves which, in contrast to central nerves, have the ability to recover from damage. Currently the therapy of choice is the use of autologous grafts where the defect is bridged with a section of autologous nerve tissue, mostly a sensory nerve [71
]. Alternatively, nerve conduits or decellularized nerve grafts can be used; however, no therapy could yield a satisfactory functional recovery [72
]. Various combinations of NTs, neuropoietic cytokines and GFLs have been shown to generate a microenvironment suitable to improve nerve repair [26
]. The results of our study may provide novel aspects for the treatment of peripheral nerve injury as the local application of a designer cytokine such as H-IL-6 with a strongly enhanced bioactivity on neuronal development and neurite outgrowth in combination with NTs and/or GFLs may create a microenvironment with a strong reparative potency.