Abstract
Glucocorticoid (GC)-induced osteoporosis has been attributed to a GC-induced suppression of pre-osteoblast proliferation. Our previous work identified a critical role for mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) in mediating the anti-proliferative effects of GCs in immortalized pre-osteoblasts, but we subsequently found that MKP-1 null mice were not protected against the pathological effects of GCs on bone. In order to reconcile this discrepancy, we have assessed the effects of GCs on proliferation, activation of the MAPK ERK1/2 and MKP-1 expression in primary adipose-derived stromal cells (ADSCs) and ADSC-derived pre-osteoblasts (ADSC-OBs). ADSCs were isolated by means of collagenase digestion from adipose tissue biopsies harvested from adult male Wistar rats. ADSC-OBs were prepared by treating ADSCs with osteoblast differentiation media for 7 days. The effects of increasing concentrations of the GC dexamethasone on basal and mitogen-stimulated cell proliferation were quantified by tritiated thymidine incorporation. ERK1/2 activity was measured by Western blotting, while MKP-1 expression was quantified on both RNA and protein levels, using semi-quantitative real-time PCR and Western blotting, respectively. GCs were strongly anti-proliferative in both naïve ADSCs and ADSC-OBs, but had very little effect on mitogen-induced ERK1/2 activation and did not upregulate MKP-1 protein expression. These findings suggest that the anti-proliferative effects of GCs in primary ADSCs and ADSC-OBs in vitro do not require the inhibition of ERK1/2 activation by MKP-1, which is consistent with our in vivo findings in MKP-1 null mice.
Highlights
Glucocorticoids (GC) are frequently used to manage diseases that result from an inappropriate inflammatory response, such as asthma, rheumatoid arthritis and inflammatory bowel syndrome
The activation of the ERK1/2 pathway is essential for proliferation in the majority of cells [15], including immortalized MBA 15.4 and MG63 pre-osteoblasts [18], and the relationship between proliferation and ERK1/2 activity in primary naïve adipose-derived stromal cells (ADSCs) and ADSC-OBs was investigated by examining ERK1/2 phosphorylation after mitogenic stimulation
The results from the present study indicate that mitogenic stimulation in primary naïve ADSCs and ADSC-derived pre-osteoblasts (ADSC-OBs) is mediated via the classical ERK1/2 pathway
Summary
Glucocorticoids (GC) are frequently used to manage diseases that result from an inappropriate inflammatory response, such as asthma, rheumatoid arthritis and inflammatory bowel syndrome. The decline in the availability of functional osteoblasts in the presence of elevated (pharmacological) levels of GCs is a consequence of GC-induced inhibition of osteoblast differentiation [5] and pre-osteoblast proliferation [5,6], an increase in osteoblast apoptosis [7] and the promotion of aberrant differentiation of osteoblast progenitor cells into an adipocytic phenotype [8] This has been demonstrated in vitro in mesenchymal stromal cells (MSCs), where low concentrations of GCs in the osteogenic differentiation cocktail (usually 10 nM dexamethasone) are sufficient for the progression towards a fully mature osteoblastic phenotype [9,10,11], while higher GC doses negatively affect proliferation [12,13] and induce an increase in triglyceride accumulation [14]. The molecular mechanism underlying the negative effects of GCs on pre-osteoblast mitogenesis have been investigated in immortalized early pre-osteoblast cell lines, there is a paucity of information regarding the molecular events that occur during the modulation of proliferation by GCs in primary MSCs and pre-osteoblasts
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