Abstract
Purpose::
To investigate possible functions of Olfactomedin 1 (Olfm1), a founding member of the family of olfactomedin domain-containing proteins that includes myocilin.
Methods::
Stably transfected rat pheochromocytoma PC12 cells were obtained by infection with a retroviral vector (control) or with a vector containing rat Olfm1 cDNA. Neuronal differentiation of PC12 cells was induced by addition of 50 ng/ml of nerve growth factor (NGF). Changes in the gene expression pattern were assessed using rat Affymetrix arrays. Quantitative RT-PCR was used to analyze changes in the expression levels of selected genes (Wif-1, annexin A1) after DNA demethylation and histone modifications induced by treatment of cells with 1 µM of 5-aza-2’-deoxycytidine for 4 days and 500 nM of Trichostatin A (TSA) for one additional day. Phalloidin staining was used to follow the reorganization of the stress fibers.
Results::
Olfm1 is expressed in the developing and adult neuronal tissues including retina. Olfm1 expression accelerated neuronal differentiation of PC12 in the presence of NGF. Olfm1-expressing cells produced 3.6 ± 0.2 neurites per cell versus 2.0 ± 0.4 neurites per control cell nine days after NGF addition. Neurites in Olfm1-expressing cells had more branches than neurites in control cells (3.3 ± 0.2 versus 1.4 ± 0.2). Olfm1 expression induced at least 2 fold changes in the levels of several hundred mRNAs compared to control cells as demonstrated by array hybridization. Several mRNAs that were highly up-regulated by Olfm1 expression (Wif1, annexin A1) have been previously shown to be inhibited in different cancers through hypermethylation of their promoters. Treatment of control cells with demethylation agents induced expression of Wif1 and annexin A1 to the levels comparable with their induction by expression of Olfm1, indicating that expression of Olfm1 may change the methylation status of several promoters. To examine possible involvement of Olfm1 in non-canonical Wnt signaling, NIH3T3 cells were treated with conditioned media from Olfm1-expressing or Wnt3a-expressing cells. In both cases, addition of conditioned media induced formation of the stress fibers within several hours. Possible involvement of Olfm1 in Wnt signaling pathways is under investigation.
Conclusions::
The action of Olfm1 may include epigenic mechanisms and suppress hypermethylation of promoters of some genes. Olfm1 may be tried as a potential theurapeutic agent in cancerogenesis.
Keywords: gene/expression • differentiation • retinal development