Gene expression profile in prion protein-deficient fibroblasts in culture

Abstract

To investigate the physiological function of the cellular isoform of prion protein (PrP(C)), the gene expression profile was studied by analyzing a cDNA expression array containing 597 clones of various functional classes in two distinct skin fibroblast cell lines designated SFK and SFH, established from PrP-deficient (PrP(-)(/-)) mice and PrP(+/+) mice, respectively. The cells were incubated in the culture medium with or without inclusion of basic fibroblast growth factor (bFGF). When SFK cells were compared with SFH cells in untreated conditions, the expression of 15 genes, including those essential for cell proliferation and adhesion, was reduced, whereas the expression of 27 genes, including those involved in the insulin-like growth factor-I (IGF-I) signaling pathway, was elevated. Northern blot analysis verified a significant down-regulation of the receptor tyrosine kinase substrate Eps8, cyclin D1, and CD44 mRNAs, and a substantial up-regulation of phosphatidylinositol 3-kinase p85, IGF-I, and serine protease inhibitor-2.2 mRNAs in SFK cells. The patterns of induction or reduction of gene expression after exposure to bFGF showed considerable overlap between both cell types. Furthermore, both Eps8 and CD44 mRNA levels were reduced greatly in the brain tissues of the cerebrum isolated from the PrP(-)(/-) mice. These results indicate that the disruption of the PrP gene resulted in an aberrant regulation of a battery of genes important for cell proliferation, differentiation, and survival, including those located in the Ras and Rac signaling pathways.

Figure 1.

Nonexpression of the PrP gene in a skin fibroblast cell line established from the PrP^−/− mice. Two cell lines were established from primary cultures of skin fibroblasts isolated from the PrP^−/− mice (SFK) or from the PrP^+/+ mice (SFH). They were maintained for 4 months in vitro before starting the experiments. The cells were incubated for 72 hours in LS medium with (SFH-B and SFK-B) or without (SFH-C and SFK-C) inclusion of 50 ng/ml of bFGF for the last 24 hours before processing for RNA preparation. Fifty nanograms of cDNA prepared from total RNA by reverse transcription (RT) was amplified by PCR using primer pairs specific for the PrP gene (lanes 1−8) or the β-actin gene (lanes 9−16) listed in Table 1 ▶ . Lanes 1 and 9 represent SFH-C cells processed for RT-PCR omitting the RT step; lanes 2 and 10, SFH-C cells processed for RT-PCR including the RT step; lanes 3 and 11, SFK-C cells processed for RT-PCR omitting the RT step; lanes 4 and 12, SFK-C cells processed for RT-PCR including the RT step; lanes 5 and 13, SFH-B cells processed for RT-PCR omitting the RT step; lanes 6 and 14, SFH-B cells processed for RT-PCR including the RT step; lanes 7 and 15, SFK-B cells processed for RT-PCR omitting the RT step; lanes 8 and 16, SFK-B cells processed for RT-PCR including the RT step. The DNA size marker (bp) is shown on the left.

Figure 2.

Southern blot hybridization of a cDNA expression array with ³²P-labeled cDNA probes prepared from mRNA isolated from SFK and SFH cells. A set of filters containing 597 arrayed mouse cDNA clones of various functional classes, immobilized as a spot of duplicate dots on a nylon membrane, were hybridized with ³²P-labeled cDNA probes prepared from poly A⁺ RNA that was isolated from bFGF-treated or untreated SFK and SFH cells maintained for 4 months in vitro: SFH-C (a), SFK-C (b), SFH-B (c), or SFK-B (d) cells. The spots indicated by arrows represent the clones of (1) the receptor tyrosine kinase substrate Eps8, (2) cyclin D1, (3) CD44, (4) phosphatidylinositol 3-kinase (PI3K) p85, (5) insulin-like growth factor (IGF-I), (6) serine protease inhibitor (Spi-2.2), and (7) β-actin.

Figure 3.

Northern blot analysis of Eps8, cyclin D1, CD44, PI3K p85, IGF-I, and Spi-2.2 mRNA expression in SFK, SFH, and SFHT cells and brain tissues isolated from the PrP^−/− and PrP^+/+ mice. A distinct skin fibroblast cell line, designated SFHT, was established from the PrP^+/− mice. SFHT cells were incubated for 72 hours in LS medium with (SFHT-B) or without (SFHT-C) inclusion of 50 ng/ml of bFGF for the last 24 hours before processing for RNA preparation. Total RNA was extracted from SFH-C, SFHT-C, SFK-C, SFH-B, SFHT-B, or SFK-B cells, and from the whole cerebral cortices isolated from the PrP^−/− mice (CBRK) or from the PrP^+/+ mice (CBRH). It was processed for Northern blot analysis by hybridization with digoxigenin (DIG)-labeled DNA probes specific for the Eps8 (a), cyclin D1 (b), CD44 (c), PI3K p85 (d), IGF-I (e), or Spi-2.2 (f) gene (upper panels), followed by rehybridization with the probe specific for the β-actin gene for standardization (lower panels) and, in limited experiments, with the probe specific for the PrP gene (lowest panels). A: Lanes 1–4 represent 2 μg of total RNA isolated from SFH-C (lane 1), SFK-C (lane 2), SFH-B (lane 3), and SFK-B (lane 4) cells, all of which were maintained for 4 months in vitro before starting the experiments. B: Lanes 1–4 represent 2 μg of total RNA isolated from the following cells: SFHT-C (lane 1), SFK-C (lane 2), SFHT-B (lane 3), and SFK-B (lane 4). SFHT cells were maintained for 4 months, whereas SFK cells were maintained for 7 months in vitro before starting the experiments. The lanes in C represent 6 μg of total RNA isolated from the brain tissues CBRH (lane 1) and CBRK (lane 2). The RNA size marker (kb) is shown in the left lane.