A Combined Proteome and Microarray Investigation of Inorganic Phosphate-induced Pre-osteoblast Cells

Abstract

Inorganic phosphate, which is generated during osteoblast differentiation and mineralization, has recently been identified as an important signaling molecule capable of altering signal transduction pathways and gene expression. A large scale quantitative proteomic investigation of pre-osteoblasts stimulated with inorganic phosphate for 24 h resulted in the identification of 2501 proteins, of which 410 (16%) had an altered abundance ratio of greater than or equal to 1.75-fold, either up or down, revealing both novel and previously defined osteoblastregulated proteins. A pathway/function analysis of these proteins revealed an increase in cell cycle and proliferation that was subsequently verified by conventional biochemical means. To further analyze the mechanisms by which inorganic phosphate regulates cellular protein levels, we undertook a mRNA microarray analysis of preosteoblast cells at 18, 21, and 24 h after inorganic phosphate exposure. Comparison of the mRNA microarray data with the 24-hour quantitative proteomic data resulted in a generally weak overall correlation; the 21-hour RNA sample showed the highest correlation to the proteomic data. However, an analysis of osteoblast relevant proteins revealed a much higher correlation at all time points. A comparison of the microarray and proteomic datasets allowed for the identification of a number of candidate proteins that are post-transcriptionally regulated by elevated inorganic phosphate, including Fra-1, a member of the activator protein-1 family of transcription factors. The analysis of the data presented here not only sheds new light on the important roles of inorganic phosphate in osteoblast function but also begins to address the contribution of post-transcriptional and post-translational regulation to a cell's expressed proteome. The ability to accurately measure changes in both protein abundance and mRNA levels on a system-wide scale represents a novel means to extract data from previously

Proteomics of spermatogenesis: from protein lists to understanding the regulation of male fertility and infertility

“…Moreover, transcriptomics poses certain limitations as the expression levels of the majority of mRNAs and proteins are not always correlated. 7,8 This limitation is particularly distinct during spermatogenesis for the following reasons: (i) male germ cell-specific proteins may be synthesized from specific alternate transcripts; (ii) transcription is arrested during spermiogenesis and pre-existing mRNAs are stored for several days; (ii) for the completion of spermatogenesis, the appropriate temporal activation of stored messages is required, and translational activation must occur independently of transcriptional control. 9,10 Therefore, it is important to explore changes in protein expression directly and systematically.…”

High Dietary Inorganic Phosphate Increases Lung Tumorigenesis and Alters Akt Signaling

“…Our previous studies found that high dietary Pi might activate the Akt pathway in the lungs of mice (8), and the activation of Akt signaling was dependent on Pi treatment in normal lung cells (15). In vitro studies indicate that Pi works as a stimulus capable of increasing or decreasing the expression of a number of pivotal genes such as those involved in the regulation of transcription, signal transduction, and cell cycle (15)(16)(17)(18)(19)(20). It is thought that excess amount of Pi intake over a long period of time is a strong factor in aging (1).…”

Phosphate (Pi)-regulated heterodimerization of the high-affinity sodium-dependent Pi transporters PiT1/Slc20a1 and PiT2/Slc20a2 underlies extracellular Pi sensing independently of Pi uptake

“…Interestingly, the Pi-dependent activation of the ERK1/2 pathway up-regulated the gene expression of the mineralization inhibitors matrix gla protein (Mgp) and osteopontin (Opn), most likely setting off a feed-back mechanism to control Pi-induced mineralization (7,16,18,19). The elevated extracellular Pi level was also shown to be important in osteoblast proliferation and differentiation (16,(20)(21)(22)(23)(24), cementoblast formation (25), odontoblast differentiation (26,27) and osteoclast differentiation (28)(29)(30).…”