Expression profiling and Ingenuity biological function analyses of interleukin-6- versus nerve growth factor-stimulated PC12 cells

Abstract

Background: The major goal of the study was to compare the genetic programs utilized by the neuropoietic cytokine Interleukin-6 (IL-6) and the neurotrophin (NT) Nerve Growth Factor (NGF) for neuronal differentiation.

Results: The designer cytokine Hyper-IL-6 in which IL-6 is covalently linked to its soluble receptor s-IL-6R as well as NGF were used to stimulate PC12 cells for 24 hours. Changes in gene expression levels were monitored using Affymetrix GeneChip technology. We found different expression for 130 genes in IL-6- and 102 genes in NGF-treated PC12 cells as compared to unstimulated controls. The gene set shared by both stimuli comprises only 16 genes.A key step is upregulation of growth factors and functionally related external molecules known to play important roles in neuronal differentiation. In particular, IL-6 enhances gene expression of regenerating islet-derived 3 alpha (REG3A; 1084-fold), regenerating islet-derived 3 beta (REG3B/PAPI; 672-fold), growth differentiation factor 15 (GDF15; 80-fold), platelet-derived growth factor alpha (PDGFA; 69-fold), growth hormone releasing hormone (GHRH; 30-fold), adenylate cyclase activating polypeptide (PACAP; 20-fold) and hepatocyte growth factor (HGF; 5-fold). NGF recruits GDF15 (131-fold), transforming growth factor beta 1 (TGFB1; 101-fold) and brain-derived neurotrophic factor (BDNF; 89-fold). Both stimuli activate growth-associated protein 43 (GAP-43) indicating that PC12 cells undergo substantial neuronal differentiation.Moreover, IL-6 activates the transcription factors retinoic acid receptor alpha (RARA; 20-fold) and early growth response 1 (Egr1/Zif268; 3-fold) known to play key roles in neuronal differentiation.Ingenuity biological function analysis revealed that completely different repertoires of molecules are recruited to exert the same biological functions in neuronal differentiation. Major sub-categories include cellular growth and differentiation, cell migration, chemotaxis, cell adhesion, small molecule biochemistry aiming at changing intracellular concentrations of second messengers such as Ca2+ and cAMP as well as expression of enzymes involved in posttranslational modification of proteins.

Conclusion: The current data provide novel candidate genes involved in neuronal differentiation, notably for the neuropoietic cytokine IL-6. Our findings may also have impact on the clinical treatment of peripheral nerve injury. Local application of a designer cytokine such as H-IL-6 with drastically enhanced bioactivity in combination with NTs may generate a potent reparative microenvironment.

Figure 1

Changes in expression of GAP-43- and REG3B mRNA levels in IL-6-stimulated PC12 cells determined by qRT-PCR versus GeneChip. Affymetrix Genechip- and qRT-PCR analyses were performed as described in the Methods section.

Figure 2

Changes in expression of GAP-43- mRNA levels in IL-6- versus NGF-stimulated PC12 cells. qRT-PCR analyses were performed as described in the Methods section.