GeneChip Microarrays Facilitate Identification of Protease Nexin-1 as a Target Gene of the Prx2 (S8) Homeoprotein

Abstract

The paired-related homeobox genes, Prx1 and Prx2, are important for normal skeletal and cardiovascular development as well as adult vascular remodeling. The identification and characterization of Prx downstream targets is crucial to understanding their function in normal developmental processes and congenital malformations. To identify Prx2 regulated genes, stably transfected NIH3T3 clones expressing Prx2 sense or antisense transcripts were generated. Expression profiles initially were established for two of the clones using Affymetrix GeneChip arrays. Over 6,400 genes were screened by the microarray approach, and approximately 500 genes differed in expression by a factor of two or more. Fifteen genes were chosen for further analysis. RT-PCR of the two transfectants used in the GeneChip analysis demonstrated that five out of the 15 genes were differentially expressed. However, after screening additional stable transfectant clones only one of the 15 genes, Protease Nexin-1 (PN-1), was differentially expressed. Subsequent Northern blot, RT-PCR, and further GeneChip analysis of additional stable transfectants confirmed that PN-1 expression is increased at least fivefold when Prx2 is overexpressed. It was demonstrated that Prx2 directly regulates PN-1 because (1) Prx2 binds to a cis element in the PN-1 promoter in vitro, and (2) Prx2 regulates the PN-1 promoter in transient transfection assays. The GeneChip analysis generated a prioritized list of other potential targets. The utility and limitations of cell culture models combined with microarray analysis for elucidating complex regulatory cascades are discussed.

Paired‐Related Homeodomain Proteins Prx1 and Prx2 are Expressed in Embryonic Pituitary Stem/Progenitor Cells and May Be Involved in the Early Stage of Pituitary Differentiation

“…Although they have many similarities and are considered to function basically as a transcription activator, an important difference is that PRX1 has three splicing variants (PRX1a, PRX1b and PRX1c), and the loss of OAR domain in PRX1b provides the property of a repressor rather than an activator (33). In addition to p21 as an indirect target of PRX2, the candidates for direct and indirect target genes of PRXs reported so far are Tenascin‐C (a vascular smooth muscle cell survival factor) (33), Matrillin‐2 (a filament‐forming protein) (34), Nexin‐1 (a broad specificity inhibitor of trypsin‐like serine proteases) (35), Collagen α1(1) (36), and Osterix (a transcription factor for osteoblast differentiation) and Runx2 (runt‐related gene 2; a key transcription factor associated with osteoblast differentiation) (37). The expression level of these genes in the pituitary and their roles in pituitary organogenesis remain obscure at present.…”

Protease Nexin-1 Inhibits Plasminogen Activation-induced Apoptosis of Adherent Cells

“…This could be the case in cells and tissues in which PN-1 is the main regulatory serpin (27) and under conditions associated with PN-1 up-regulation. For instance, the recently demonstrated up-regulation of the PN-1 gene by the Prx2 homeoprotein suggests that this serpin might be involved in skeletal and cardiovascular development as well as in adult vascular remodeling (73). Increased expression of PN-1 in tumor cell lines also suggests its involvement in tumorigenesis (74,75).…”

The Effects of Crude Oil on Skin Histology of Poecilia sphenops (Valenciennes, 1846) (Cyprinidae, Teleostei)

“…Ham petrol içeriğinde bulunan poliaromatik hidrokarbonların çeşitli biyolojik süreçlerdeki olumsuz etkilerine ilişkin çok sayıda araştırma vardır [3][4][5][6][7][8][9][10][11][12] .…”