Phosphodiesterase 10A Is a Mediator of Osteogenic Differentiation and Mechanotransduction in Bone Marrow-Derived Mesenchymal Stromal Cells

Abstract

Bone marrow-derived mesenchymal stromal cells (hMSCs) are capable of differentiating into the osteogenic lineage, and for osteogenic differentiation, mechanical loading is a relevant stimulus. Mechanotransduction leads to the formation of second messengers such as cAMP, cGMP, or Ca²⁺ influx resulting in the activation of transcription factors mediating gene regulation. The second messengers cAMP and cGMP are degraded by phosphodiesterase isoenzymes (PDE), but the role of these enzymes during osteogenic differentiation or mechanotransduction remains unclear. Here, we focused on the isoenzyme phosphodiesterase 10A (PDE10A) and its role during osteogenic commitment and mechanotransduction. We observed a time-dependent decrease of PDE10A expression in hMSC undergoing differentiation towards the osteogenic lineage. PDE10A inhibition by papaverine diminished osteogenic differentiation. While applying mechanical strain via cyclic stretching of hMSCs led to an upregulation of PDE10A gene expression, inhibition of PDE10A using the drug papaverine repressed expression of mechanoresponsive genes. We conclude that PDE10A is a modulator of osteogenic differentiation as well as mechanotransduction in hMSCs. Our data further suggests that the relative increase of cAMP, rather than the absolute cAMP level, is a key driver of the observed effects.

Figure 1

PDE10A expression in human primary MSC (hMSC, n = 7), in human neuroblastoma SH-SY5Y cells (n = 9), in murine primary MSC (mMSC, n = 3), in murine MC3T3 cells (n = 3), and in murine brain lysates (n = 3) is shown. Murine MSC and brain lysates were prepared from the identical mice. RPS27A (human samples) and B2m (murine samples) were used as housekeeping genes. QPCR data were obtained from technical triplicates, and results are shown as mean ± SEM; fold change was calculated with the ΔΔCT method.

Figure 2

PDE10A expression in hMSC after osteogenic differentiation. QPCR (a) and Western blot (b) analysis of PDE10A as well as the expression of the osteogenic markers RUNX2 and ALPL (c) in primary human MSC (n = 4) differentiated towards the osteogenic lineage for different time points as indicated. RPS27A was used as the housekeeping gene; β-actin was used as loading control. QPCR data were obtained from technical triplicates, and results are shown as mean ± SEM. Fold change was calculated with the ΔΔCT method. Significances were calculated with the Student t-test (^∗p < 0.05; ^∗∗p < 0.005). A representative Western blot is shown. ALPL: tissue nonspecific alkaline phosphatase; c: control; oD: osteogenic differentiation; PDE10a: phosphodiesterase 10a; RUNX2: runt-related transcription factor 2.

Figure 3

Effect of papaverine on viability and apoptosis of hMSCs derived from four donors. Cells were treated with 0, 1, 10, and 100 μM papaverine, and viability (a) and apoptosis assays (b) were performed 24, 48, and 72 h later. Relative luminescence is given. Data are expressed as mean of two independent experiments ± SEM and normalized to untreated control. Each measurement was performed in technical triplicate. Student's t-test was used for statistical analysis (^∗p < 0.005).

Figure 4

Analysis of osteogenic differentiation in primary human MSC (n = 5). (a) QPCR analyses of SPP1, RUNX2, and ALPL expression after 14 days of osteogenic differentiation and respective controls with and without 10 μM papaverine or 1 mM db-cAMP. RPS27A was used as the housekeeping gene. QPCR data were obtained from technical triplicates and results are shown as mean ± SEM. Fold change was calculated with the ΔΔCT method. Significances were calculated with the Student t-test (^∗p < 0.05; ^∗∗p < 0.005). (b) Alizarin red staining of primary MSC differentiated towards the osteogenic lineage for 14 days with and without application of 10 μM papaverine or 1 mM db-cAMP. Four representative donors are shown. The bar represents 100 μm. ALPL: tissue nonspecific alkaline phosphatase; c: control; db-cAMP: dibutyryl-cAMP; oD: osteogenic differentiation; Papa: papaverine; RUNX2: runt-related transcription factor 2; SPP1: secreted phosphoprotein 1.

Figure 5

Gene expression after cyclic stretching of primary MSCs derived from 13 donors. Relative mRNA expression of PDE10A and the mechanoresponsive genes FOS and PTGS2 15 min and 4 h after mechanical loading. QPCR data were obtained from three independent qPCR experiments. Results are shown as mean ± SEM; fold change was calculated with the ΔΔCT method and normalized to basal activity (nonstretched, dashed line). RPS27A served as the housekeeping gene. Significances were calculated with the Student t-test (^∗p < 0.05). FOS: fos protooncogene; PDE10A: phosphodiesterase 10A; PTGS2: prostaglandin-endoperoxide synthase 2.

Figure 6

Effect of PDE10A inhibition and cAMP stimulation on mechanotransduction in primary MSC. Relative mRNA expression of the mechanoresponsive genes PTGS2 and FOS 15 min after cyclic stretching and PDE10A 4 h after cyclic stretching. Cells were pretreated with 10 μM papaverine for 24 h or pretreated with 1 mM db-cAMP for 1 h. Results are shown as mean of five independent experiments by using five different MSC donors ± SEM and normalized to the values obtained from stretched samples. Fold change was calculated with the ΔΔCT method, and RPS27A served as the housekeeping gene. Significances were calculated with the Student t-test (^∗p < 0.05; ^∗∗p < 0.005). cs: cyclic stretching; db-cAMP: dibutyryl-cAMP; FOS: fos protooncogene; Papa: papaverine; PDE10A: phosphodiesterase 10A; PTGS2: prostaglandin-endoperoxide synthase 2.