Detection of a proliferation specific gene during development of the osteoblast phenotype by mRNA differential display

Abstract

Fetal rat calvarial-derived osteoblasts in vitro (ROB) reinitiate a developmental program from growth to differentiation concomitant with production of a bone tissue-like organized extracellular matrix. To identify novel genes which may mediate this sequence, we isolated total RNA from three stages of the cellular differentiation process (proliferation, extracellular matrix maturation, and mineralization), for screening gene expression by the differential mRNA display technique. Of 15 differentially displayed bands that were analyzed by Northern blot analysis, one prominent 310 nucleotide band was confirmed to be proliferation-stage specific. Northern blot analysis showed a 600-650 nt transcript which was highly expressed in proliferating cells and decreased to trace levels after confluency and throughout the differentiation process. We have designated this transcript PROM-1 (for proliferating cell marker). A full length PROM-1 cDNA of 607 bp was obtained by 5' RACE. A short open reading frame encoded a putative 37 amino acid peptide with no significant similarity to known sequences. Expression of PROM-1 in the ROS 17/2.8 osteosarcoma cell line was several fold greater than in normal diploid cells and was not downregulated when ROS 17/2.8 cells reached confluency. The relationship of PROM-1 expression to cell growth was also observed in diploid fetal rat lung fibroblasts. Hydroxyurea treatment of proliferating osteoblasts blocked PROM-1 expression; however, its expression was not cell cycle regulated. Upregulation of PROM-1 in response to TGF-beta paralleled the stimulatory effects on growth as quantitated by histone gene expression. In conclusion, PROM-1 represents a small cytoplasmic polyA containing RNA whose expression is restricted to the exponential growth period of normal diploid cells; the gene appears to be deregulated in tumor derived cell lines.

Effects of low temperature and lactate on osteogenic differentiation of human amniotic mesenchymal stem cells

“…During development, preosteoblasts underwent a series of differentiation stages, such as growth, matrix development, and matrix mineralization [10]. In preosteoblasts, cell differentiation occurred when tissue-specific transcriptional switches repressed proliferation [10,29,30]. It has been shown that osteogenic differentiation contributed, in part, to the cessation of cell growth [9,31].…”

Differential Expression of Neuroleukin in Osseous Tissues and Its Involvement in Mineralization During Osteoblast Differentiation

“…For the study of differentiation, cells were plated at 10 6 cells/100-mm dish and differentiation was induced at time of plating (day 0) by addition of 50 g/ml of ascorbic acid and 10 mM of ␤-glycerol phosphate to the medium. Medium supplemented with these inducing agents was changed every 3 days, and cells from culture days 1,2,4,7,10,13,16,19,22,25,28, and 31 were harvested and examined. Each time-point contains two dishes.…”

“…More specifically, MC3T3-E1 osteoblast-like cells have been studied widely with respect to proliferation, bone matrix accumulation, mineralization, growth factor regulation, changes in morphology and metabolism, signal transduction, and gene expression. (3)(4)(5)(6)(7) To examine the process of osteoblast differentiation and to search for molecules that might play significant regulatory roles in the transition from one phase to another, differential messenger RNA (mRNA) analysis (8) was performed on RNA isolated from numerous time points during the progression of MC3T3-E1 cells from their initial proliferative state to matrix accumulation and mineralization. Using this approach, we identified a number of known and novel differentially expressed complementary DNAs (cDNAs), one of which was identified as neuroleukin (NLK).…”

Cloning and characterization of osteoactivin, a novel cDNA expressed in osteoblasts

“…A number of different techniques such as differential display [Ryoo et al, 1997; Xu et al, 2000], subtractive hybridization [Petersen et al, 2000], and, most recently, gene array analysis [Beck et al, 2001] have led to the identification of novel factors that regulate bone cell development and function. Among these factors are OF45 [Petersen et al, 2000], connective tissue growth factor (CTGF) [Xu et al, 2000], the TGF‐β superfamily members, lefty‐1 [Seth et al, 2000], best‐5 [Grewal et al, 2000] and PROM‐1 [Ryoo et al, 1997], murine osteoclast inhibitory lectin (mOCIL) [Zhou et al, 2001], and RANK‐L [Horowitz et al, 2001; Teitelbaum, 2000]. Some of these factors promote osteoblast differentiation and enhance osteogenesis while others are produced by osteoblasts and regulate osteoclast development and/or function.…”