Abstract
BackgroundOsteogenic induction and bone formation are heavily affected by environmental factors, including estrogen, estrogen receptors, and coregulatory proteins, such as the recently reported proline-, glutamic acid-, and leucine-rich protein 1(Pelp1).ObjectiveTo investigate Pelp1 expression in rat bone mesenchymal stem cells (rBMSCs) during cell proliferation and osteogenic differentiation.MethodsrBMSCs were cultured in routine and osteogenic differentiation media. Cell proliferation was assessed at days 1, 3, 5, 7, 9, 11, 14, and 21. Pelp1 protein expression in the nucleus and cytoplasm were detected by immunocytochemical analysis. Real-time RT-PCR and western blot were used to detect mRNA and protein expressions of Pelp1, osteocalcin (OCN), and alkaline phosphatase (ALP).ResultsOver 21 days, rBMSCs in routine culture exhibited a 1-2 day lag phase and exponential growth from day 3 to 9, plateauing at day 9, and correlated with temporal mRNA expression of Pelp1, which almost reached baseline levels at day 21. In osteogenic induction cultures, Pelp1 mRNA levels rose at day 9 and steadily increased until day 21, reaching 6.8-fold greater value compared with day 1. Interestingly, Pelp1 mRNA expression in osteogenic cultures exhibited a trend similar to that of OCN expression. Pelp1 knockdown by siRNA transfection inhibited undifferentiated rBMSC proliferation, and bone markers OCN and ALP expressions in rBMSCs cultured in routine and osteogenic differentiation media.ConclusionsPelp1 may be a key player in BMSCs proliferation and osteogenic differentiation, meriting further consideration as a target for development of therapies for pathological bone loss conditions, such as menopausal bone loss.
Highlights
The metabolic roles of estrogen binding to estrogen receptors (ERs) have been extensively documented in a variety of cells and tissues [1,2,3], but their role in osteoblast cell lines has only recently been reported as an important factor in overall bone health [4]
RBMSCs were split at day 5 to prevent them from reaching confluence at day 7, and Pelp1 expression was assessed at days 7, 9, 11 and 14
Results from the present study suggest that undifferentiated rat bone mesenchymal stem cells (rBMSCs), which are cells targeted by estrogen and involved in bone development, express Pelp1 in their cytoplasm and nuclei, but mainly in their cytoplasm, suggesting a decreased osteogenesis since Pelp1 has to localize to the nucleus to exert its genomic effects [9,10,20]
Summary
The metabolic roles of estrogen binding to estrogen receptors (ERs) have been extensively documented in a variety of cells and tissues (such as brain, breast, cardiovascular system, and uterus) [1,2,3], but their role in osteoblast cell lines has only recently been reported as an important factor in overall bone health [4]. A better understanding of the roles of estrogen, ERs, and regulatory cofactors in osteogenic processes may form a basis for therapeutic improvements, as well as for the development of novel therapies against menopausal bone loss. Expression of the ER proline-, glutamic acid-, and leucine-rich protein 1 (Pelp1) has been reported in the nucleus and cytoplasm of a wide variety of tissues, most notably the brain [8], mammary gland, ovaries, and uterus [9,10]. Osteogenic induction and bone formation are heavily affected by environmental factors, including estrogen, estrogen receptors, and coregulatory proteins, such as the recently reported proline-, glutamic acid-, and leucine-rich protein 1(Pelp). Pelp knockdown by siRNA transfection inhibited undifferentiated rBMSC proliferation, and bone markers OCN and ALP expressions in rBMSCs cultured in routine and osteogenic differentiation media. Conclusions: Pelp may be a key player in BMSCs proliferation and osteogenic differentiation, meriting further consideration as a target for development of therapies for pathological bone loss conditions, such as menopausal bone loss
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