Cyclin‐dependent kinase 8/19 inhibition suppresses osteoclastogenesis by downregulating RANK and promotes osteoblast mineralization and cancellous bone healing

Abstract

Cyclin‐dependent kinase 8 (CDK8) is a mediator complex‐associated transcriptional regulator that acts depending on context and cell type. While primarily under investigation as potential cancer therapeutics, some inhibitors of CDK8—and its paralog CDK19—have been reported to affect the osteoblast lineage and bone formation. This study investigated the effects of two selective CDK8/19 inhibitors on osteoclastogenesis and osteoblasts in vitro, and further evaluated how local treatment with a CDK8/19 inhibitor affects cancellous bone healing in rats. CDK8/19 inhibitors did not alter the proliferation of neither mouse bone marrow–derived macrophages (BMMs) nor primary mouse osteoblasts. Receptor activator of nuclear factor κΒ (NF‐κB) ligand (RANKL)‐induced osteoclastogenesis from mouse BMMs was suppressed markedly by inhibition of CDK8/19, concomitant with reduced tartrate‐resistant acid phosphatase (TRAP) activity and C‐terminal telopeptide of type I collagen levels. This was accompanied by downregulation of PU.1, RANK, NF‐κB, nuclear factor of activated T‐cells 1 (NFATc1), dendritic cell‐specific transmembrane protein (DC‐STAMP), TRAP, and cathepsin K in RANKL‐stimulated BMMs. Downregulating RANK and its downstream signaling in osteoclast precursors enforce CDK8/19 inhibitors as anticatabolic agents to impede excessive osteoclastogenesis. In mouse primary osteoblasts, CDK8/19 inhibition did not affect differentiation but enhanced osteoblast mineralization by promoting alkaline phosphatase activity and downregulating osteopontin, a negative regulator of mineralization. In rat tibiae, a CDK8/19 inhibitor administered locally promoted cancellous bone regeneration. Our data indicate that inhibitors of CDK8/19 have the potential to develop into therapeutics to restrict osteolysis and enhance bone regeneration.

Characterizing CDK8/19 Inhibitors through a NFκB-Dependent Cell-Based Assay

“…All the thienopyridines exhibited strong inhibitory activities in the 293-WT-NFκB-Luc cell-based assay with IC 50 values ranging from 4.1 nM to 50.6 nM and plateau inhibition of ~80% (Figure 4B). Interestingly, the IC 50 values measured in this assay were very highly correlated with the values of EC200 (the concentration enhancing ALPase activity to 200% of the control) in the ALPase assay measured by Saito et al [16] (R 2 = 0.98), providing a strong indication that the in vitro bone anabolic activity is most likely mediated through CDK8/19 inhibition, in agreement with Amirhosseini et al [17]. In addition, the inhibitory activities of 15k, 15u and 15w were also tested in 293-dKO-NFκB-Luc cells and none of them showed any activity in these cells (Figure 4C), demonstrating that NFκB inhibition by these compounds was mediated through CDK8/19.…”

“…Perhaps the best demonstration of the reliability of this assay appears in Figure 4C, where the IC 50 values measured for six thienopyridine derivatives showed a near-perfect correlation with EC200 values that were determined by another group, in an entirely different assay, using different batches of the compounds. The results obtained in this analysis demonstrated CDK8/19-inhibitory activity for all six thienopyridines, most of which have not been previously tested for this activity, and it confirms the report that the bone anabolic activity is mediated by CDK8/19 inhibition [17]. The latter paper identified several factors affected by CDK8/19 inhibitors in bone marrow-derived cells and, remarkably, NFκB was one of these factors [17].…”

“…The results obtained in this analysis demonstrated CDK8/19-inhibitory activity for all six thienopyridines, most of which have not been previously tested for this activity, and it confirms the report that the bone anabolic activity is mediated by CDK8/19 inhibition [17]. The latter paper identified several factors affected by CDK8/19 inhibitors in bone marrow-derived cells and, remarkably, NFκB was one of these factors [17].…”

“…A recent publication reported that a thienopyridine derivative (15w) is a selective CDK8/19 inhibitor that (along with senexin B) promotes osteoblast mineralization and bone regeneration [17]. 15w is one of a series of compounds that were originally discovered and optimized for in vitro bone anabolic activity using an alkaline phosphatase (ALPase) activity assay in a mouse bone marrow stromal cell line ST2 [16].…”

“…The WT reporter cells provided a sensitive and robust assay for CDK8/19 inhibition of NFκB-induced transcription; whereas, the matching dKO reporter cells offered a selectivity control for CDK8/19 dependence of the effects of tested inhibitors. Using this assay, we have characterized different CDK8/19-inhibiting small molecules, including a series of thienopyridines with in vitro bone anabolic activity [16], one of which has been recently identified as a selective CDK8/19 inhibitor [17], as well as several inhibitors of other CDKs. Our results demonstrate a striking correlation between the effects on CDK8/19 and bone anabolic activity and provide a comparison of the effects of CDK8/19 inhibitors and inhibitors of some other CDKs on NFκB activity.…”