Differentiation dependent expression of urocortin's mRNA and peptide in human osteoprogenitor cells: influence of BMP-2, TGF-beta-1 and dexamethasone

Abstract

Urocortin-1 (UCN) a corticotropin releasing-factor (CRF) related peptide, has been found to be expressed in many different tissues like the central nervous system, the cardiovascular system, adipose tissue, and skeletal muscle. The effects of UCN are mediated via stimulation of CRF-receptors 1 and 2 (CRFR1 and 2, CRFR's) with a high affinity for CRFR2. It has been shown that the CRF-related peptides and CRFR's are involved in the regulation of stress-related endocrine, autonomic and behavioural responses. Using immunocytochemistry, immunohistochemistry and RT-PCR, we now can show the differentiation dependent expression of UCN mRNA and peptide in human mesenchymal progenitor cells (MSCs) directed to the osteoblastic phenotype for the first time. UCN expression was down regulated by TGF-beta and BMP-2 in the early proliferation phase of osteoblast development, whereas dexamethasone (dex) minimally induced UCN gene expression during matrix maturation after 24 h stimulation. Stimulation of MSCs for 28 days with ascorbate/beta-glycerophosphate (asc/bGp) induced UCN gene expression at day 14. This effect was prevented when using 1,25-vitamin D3 or dex in addition. There was no obvious correlation to osteocalcin (OCN) gene expression in these experiments. In MSCs from patients with metabolic bone disease (n = 9) UCN gene expression was significantly higher compared to MSCs from normal controls (n = 6). Human MSCs did not express any of the CRFR's during differentiation to osteoblasts. Our results indicate that UCN is produced during the development of MSCs to osteoblasts and differentially regulated during culture as well as by differentiation factors. The expression is maximal between proliferation and matrix maturation phase. However, UCN does not seem to act on the osteoblast itself as shown by the missing CRFR's. Our results suggest new perspectives on the role of urocortin in human skeletal tissue in health and disease.

Fig. 1

Gene expression of UCN by PCR in human mesenchymal progenitor cells (MSCs) directed to the osteoblastic phenotype after incubation with bGp/asc (upper picture). Under the same culture conditions the MSCs die not express any CRFRs. Human heart cells were used as positive control, because of known expression of UCN and CRFRs (lower picture)

Fig. 2

a Gene expression of UCN in primary human osteoblasts after incubation at confluence with bGp/asc/dex (* P < 0.05 vs. day 4, 7 and 28). Mean ± SEM (n = 5) analysed by RT–PCR, as described in methods. b A comparison between the differentiation model as proposed by Owen in rat osteoblasts and confirmed by Siggelkow in the human system and UCN gene expression after stimulation with bGp/asc/dex shows that the maximum of these expression occurred in the matrix maturation stage. It is important to mention that this is a schematic comparison for better understanding the differentiation phase of urocortin-expression

Fig. 3

Gene expression by real-time PCR of urocortin (UCN, a–d) and osteocalcin (OCN, e–h) in MSCs stimulated for 28 days beginning 3 days after confluence. Control cells were fed with 1% human serum. For stimulation ascorbate (asc) and beta-glycerophoshate (bGp) were used either alone (a, e), or in combination with 1,25-vitamin D3 (D3, b, f) or dexamethasone (dex, c, g) or D3 and dex (d, h). UCN and OCN are depicted relative to the housekeeping gene beta-actin. Values at day 0 were arbitrarily put to 1, therefore the presented data show relative changes in gene expression. Differences between control and stimulated cultures were analysed by Kruskal–Wallis test (* P < 0.05, ** P < 0.01)

Fig. 4

Immunocytochemistry, immunofluorecsence and DAPI staining (400× magnification). Osteoblasts in the cell culture stained positive for UCN after stimulation with bGp/asc/dex. a Control cells (unstained), b Immunocytochemically stained cells for UCN (day 4). c The arrows show the stained cytoplasma of cells in the same culture. d, g UCN shows a cytoplasmic staining in the immunofluorecsence (IF) analysis (D = day 14, G = day 28). e, h Interestingly, UCN shows also an inhomogeneous nuclear staining with differing intensities in some cells (DAPI staining, E = day 14, H = day 28). Bars = 27 μm. F, I: To proof the specificity of this finding, the anti-UCN antibodies were incubated over-night with synthetic human UCN prior to application in IF (F = day 14, I = day 28). This pre-treatment completely eliminated the UCN signal, confirming the specificity of our UCN detection method. j–l Renal proximal tubuli were stained for UCN as positive controls (confocal microscopy)

Fig. 5

Gene expression of UCN in MSCs after 24 h incubation on day 4, day 14 and day 28 with single osteogenic factors [TGF-beta-1 (1 ng/ml), BMP-2 (50 ng/ml), or dexamethasone (dex) (10⁻⁸ M)] compared to control (0.1% BSA with solvent). Mean ± SEM (n = 4) analyzed by RT–PCR, as described in methods. Values were normalized to the housekeeping gene EEF1A1 and presented as percentage of the maximum expression of each gene. a—P < 0.01, b—P < 0.05 and c—P < 0.01 versus control are significant as calculated by Kruskal–Wallis-test. There is a decrease in basal expression of UCN from day 4 to day 28 (white bar). At day 4 the inhibition of UCN expression by TGF-beta and BMP-2 is highly significant, the effect is less on day 14 and not detectable late in culture. In contrast, dex had no effect early at day 4, but an increase beginning at day 14 with the maximum effect on day 28

Fig. 6

Real-time PCR measuring the expression of UCN in relation to a housekeeping gene (beta-actin) in primary cultures of MSCs established from patients with metabolic bone diseases (n = 9) and healthy controls (n = 6), grown under standard conditions without differentiation factors. Differences between the two groups were analysed by Kruskal–Wallis test (* P < 0.05)