Transcriptional regulation induced by cAMP elevation in mouse Schwann cells

Abstract

In peripheral nerves, Schwann cell development is regulated by a variety of signals. Some of the aspects of Schwann cell differentiation can be reproduced in vitro in response to forskolin, an adenylyl cyclase activator elevating intracellular cAMP levels. Herein, the effect of forskolin treatment was investigated by a comprehensive genome-wide expression study on primary mouse Schwann cell cultures. Additional to myelin-related genes, many so far unconsidered genes were ascertained to be modulated by forskolin. One of the strongest differentially regulated gene transcripts was the transcription factor Olig1 (oligodendrocyte transcription factor 1), whose mRNA expression levels were reduced in treated Schwann cells. Olig1 protein was localized in myelinating and nonmyelinating Schwann cells within the sciatic nerve as well as in primary Schwann cells, proposing it as a novel transcription factor of the Schwann cell lineage. Data analysis further revealed that a number of differentially expressed genes in forskolin-treated Schwann cells were associated with the ECM (extracellular matrix), underlining its importance during Schwann cell differentiation in vitro. Comparison of samples derived from postnatal sciatic nerves and from both treated and untreated Schwann cell cultures showed considerable differences in gene expression between in vivo and in vitro, allowing us to separate Schwann cell autonomous from tissue-related changes. The whole data set of the cell culture microarray study is provided to offer an interactive search tool for genes of interest.

Figure 1. Differential gene expression upon forskolin treatment

The differential expression of the strongest increased (A) and decreased (B) gene transcripts was validated by qRT–PCR in treated compared with untreated primary mouse Schwann cells. Data were normalized to the expression of 60s. The columns represent the mean value of 12 experimental samples, and the error bars indicate the S.D. *: P≤0.005, **: P≤0.0001, ***: P≤0.00001. Raw data are provided as Supplementary Table S3 (available at http://www.asnneuro.org/an/006/an006e142add.htm).

Figure 2. Expression of Olig1 in sciatic nerves and cultured Schwann cells

Immunofluorescent stainings of transversal (A) and longitudinal (C) tissue sections of sciatic nerves from P7 mice revealed Olig1 immunofluorescence in Remak bundles (arrows), identified by a bundle of unmyelinated (MBP-negative) small diameter axons, and in a subset of myelinating Schwann cells (arrowheads). (B) Remak bundle localization was confirmed by the expression of p75^NTR. (D) A progressive increase of Olig1 mRNA expression levels was detected during peripheral nerve development by qRT–PCR. Data were normalized to the expression of 60s, and values at P0 were set to 1. Each data point represent the mean value of at least eight experimental samples, and the error bars indicate the S.D. (E) In vitro, Olig1 expression was predominantly detected in the cytoplasm of cultured Schwann cells (E, arrows), whereas nuclear staining was significantly weaker (E, arrowheads). NF: neurofilament. Bar: A: 100 μm; B, C, E: 20 μm.

Figure 3. GO-annotations of differentially expressed genes due to forskolin treatment

Analysis of molecular functions revealed that several transcripts increased with forskolin are associated with cytoskeletal protein and actin binding. Both sets of transcripts either increased or decreased due to forskolin manifested association with the cellular component of ECM and with the plasma membrane. The term of basement membrane and integrin complex was exclusively enriched in forskolin-reduced transcripts. GO-annotation analysis of transcripts decreased with forskolin showed an enrichment of genes implicated in intracellular signaling cascade and in the MAPKK cascade. Raw data are provided as Supplementary Table S4 (available at http://www.asnneuro.org/an/006/an006e142add.htm).

Figure 4. Effect of forskolin treatment on mRNA expression levels of known components of the ECM in Schwann cells

(A) Data analysis of the microarray was performed by a two-way ANOVA, and unadjusted P-values <0.01 were accounted as significant. Forskolin had a regulatory effect on the majority of investigated ECM-associated genes. n.s.: not significant. (B) Schematic illustration of components of the ECM and the basal lamina in Schwann cells. The effect of forskolin on gene expression of selected genes associated with the ECM and the basal lamina (red line) was shown by blue (reduced), red (increased) or green (unaltered) arrows.

Figure 5. Comparison analysis of gene expression between primary mouse Schwann cell cultures and developing sciatic nerve samples

(A, B) Analysis of the whole-genome revealed distinct gene transcription between in vivo and in vitro, illustrated by PCA (A). A well-defined cluster can be depicted of developing nerve samples, whereas P60 form an individual cluster. Distinct expression between primary Schwann cell cultures and in vivo samples could be confirmed by a heat map analysis, indicated by the dendrogram (B). (C, D) Analysis based on only forskolin-dependent differentially expressed transcripts resulted in distinct clusters between treated and untreated Schwann cells, as well as between in vivo and in vitro (C). Heat map analysis revealed that forskolin-treated Schwann cells associate within the same branch as the samples derived from the nerve tissues (D, arrow).