Influence of ARHGEF3 and RHOA Knockdown on ACTA2 and Other Genes in Osteoblasts and Osteoclasts

Osteoporosis is a common bone disease that has a strong genetic component. Genome-wide linkage studies have identified the chromosomal region 3p14-p22 as a quantitative trait locus for bone mineral density (BMD). We have previously identified associations between variation in two related genes located in 3p14-p22, ARHGEF3 and RHOA, and BMD in women. In this study we performed knockdown of these genes using small interfering RNA (siRNA) in human osteoblast-like and osteoclast-like cells in culture, with subsequent microarray analysis to identify genes differentially regulated from a list of 264 candidate genes. Validation of selected findings was then carried out in additional human cell lines/cultures using quantitative real-time PCR (qRT-PCR). The qRT-PCR results showed significant down-regulation of the ACTA2 gene, encoding the cytoskeletal protein alpha 2 actin, in response to RHOA knockdown in both osteoblast-like (P<0.001) and osteoclast-like cells (P = 0.002). RHOA knockdown also caused up-regulation of the PTH1R gene, encoding the parathyroid hormone 1 receptor, in Saos-2 osteoblast-like cells (P<0.001). Other findings included down-regulation of the TNFRSF11B gene, encoding osteoprotegerin, in response to ARHGEF3 knockdown in the Saos-2 and hFOB 1.19 osteoblast-like cells (P = 0.003– 0.02), and down-regulation of ARHGDIA, encoding the Rho GDP dissociation inhibitor alpha, in response to RHOA knockdown in osteoclast-like cells (P<0.001). These studies identify ARHGEF3 and RHOA as potential regulators of a number of genes in bone cells, including TNFRSF11B, ARHGDIA, PTH1R and ACTA2, with influences on the latter evident in both osteoblast-like and osteoclast-like cells. This adds further evidence to previous studies suggesting a role for the ARHGEF3 and RHOA genes in bone metabolism

Substitution of peat moss with softwood biochar for soil-free marigold growth

Peat moss has historically been a key component of soil-free substrates in the greenhouse and nursery industries. However, the increasing expense of peat, negative impacts of peat mining on wetland ecosystems, and growing perception of peat as unsustainable have led to investigation for alternatives. Biochar (BC) is a promising substitute for peat, yet the majority of studies examine additions of BC to peat-based substrates rather than replacing the peat component or employ relatively low substitution rates. Furthermore, at high substitution rates the alkalinity common to many BCs may increase substrate pH and adversely impact plant production. We evaluated BC substitution for peat and pH adjustment of resulting substrates on marigold (Tagetes erecta L.) performance under standard greenhouse conditions. A high pH (10.9) softwood BC (800 °C) was substituted for peat in a standard 70:30 (v/v) peat:perlite mixture at 10% total volume increments. Substrate pH was either not adjusted or adjusted to pH 5.8 using a BC by-product, pyroligneous acid (PLA). Germination was inhibited in pH adjusted substrates with high BC substitution (50–70% total substrate volume) likely due to higher dosages of PLA needed to neutralize pH. At harvest (flowering stage, 9 weeks) the initial pH gradient (4.4–10.4) in substrates that were not pH adjusted had converged to pH 5.6–7.5, and BC substitution for peat did not negatively impact marigold biomass or flowering. At low substitution rates (10–30% total substrate volume), marigold biomass and leaf SPAD values were greater than the control peat-perlite mixture (0% BC). This study demonstrates that softwood BC can be considered as a full replacement for peat in soil-free substrates, and even at high rates (70% total substrate volume) does not require pH adjustment for marigold production. Crop- and BC-specific considerations and economic potential should be investigated for wider application