Effects of transient PTH on early proliferation, apoptosis, and subsequent differentiation of osteoblast in primary osteoblast cultures

Abstract

In primary calvarial osteoblast cultures derived from transgenic mice expressing green fluorescent protein (GFP) under the control of 3.6-kb Col1a1 promoter, the emergence of GFP signal marks the transition of multipotential osteoprogenitors into preosteoblasts. Early transient treatment (days 1-7) of these cultures with parathyroid hormone (PTH) has an anabolic effect that is not associated with an increase in total DNA content or cell number in day 21 cultures. In the present study, the effect of early PTH treatment on cell proliferation and apoptosis was examined in greater detail in GFP(+) and GFP(-) cells using flow cytometry. In preconfluent cultures, PTH significantly reduced the proportion of cells in S phase but increased those in G(0)/G(1) and G(2)+M phases in both GFP(+) and GFP(-) subpopulations. PTH decreased apoptosis only in GFP(-) but not GFP(+) cells, indicating an increased survival of GFP(-) cells. In contrast, PTH did not change the amounts of cell proliferation and apoptosis seen in either compartment after these cultures reached confluence. To further assess the effect of early PTH treatment on osteogenic differentiation, secondary cultures of sorted GFP(+) or GFP(-) cells were obtained from day 7 primary cultures that had been treated for 1 wk with PTH. This treatment resulted in larger areas of GFP expression accompanied by increased xylenol orange/von Kossa staining in the secondary cultures of GFP fractions. Early transient PTH treatment appears to enhance the commitment of progenitor cells to an osteogenic fate and results in a higher proportion of cells that achieve full osteoblast differentiation.

Nmp4/CIZ Suppresses the Parathyroid Hormone Anabolic Window by Restricting Mesenchymal Stem Cell and Osteoprogenitor Frequency

“…It is the accumulation of repeated new waves of osteoprogenitors with enhanced osteogenic potential that mediates the PTHstimulated increase in bone mass [28,32]. This may also explain that the observed lag period before the enhanced PTH-induced bone formation phase is initiated in the Nmp4-null mice.…”

“…PTH did not significantly impact the number of CFU-F AlkPhos + colonies recovered from the BM of either genotype, although there was a trend toward modestly elevating the frequency of these cells. A variety of studies have shown conflicting stimulatory and inhibitory effects of PTH on osteoprogenitor proliferation [28][29][30]; however, the WT and null mice were treated with intermittent PTH or vehicle for 3 weeks (number of mice/experimental group = 10-14). A 2-factor ANOVA was used to evaluate the impact of genotype and treatment on the individual parameters.…”

“…This may also explain that the observed lag period before the enhanced PTH-induced bone formation phase is initiated in the Nmp4-null mice. If indeed the anabolic effect of intermittent PTH is the result of consecutive waves of committed osteoblast differentiation accumulated from each PTH exposure, in which hormone only acts on the BM early osteoprogenitor cells [28], then the rate of PTH osteoprogenitor recruitment would be equivalent in both the WT and KO mice, but the WT osteoprogenitor pool would be depleted before the KO population. This is consistent with the observed equivalent addition of bone during the first 2 weeks of treatment, but the divergence in both serum osteocalcin and bone formation in the null mice at 3 weeks [7 and the present work].…”

The osteocyte as a therapeutic target in the treatment of osteoporosis

“…The PTH stimulates bone formation by increasing the number of osteoblasts [Wang et al 2007]. The PTH effects are mediated by a G-protein-coupled receptor, the PTH receptor 1 [Maeda et al 2013;Van Der Lee et al 2013].…”

Intermittent PTHrP(1–34) Exposure Augments Chondrogenesis and Reduces Hypertrophy of Mesenchymal Stromal Cells

“…However, constant application of PTHrP in chondrogenic medium suppressed chondrogenesis instead of selectively inhibiting hypertrophic features only [6,18]. Interestingly, PTH can exert opposing effects on the osteogenic differentiation of osteoblasts [19,20], rat MSCs [21], and on bone mass dependent on a constant versus intermittent application regime. Whereas constant application acted catabolically on bone, an intermittent application (eg, once daily) provided anabolic effects, resulting in a net increase in bone mass [22,23].…”