CDK12 mediates the inhibition effect of mechanical unloading on osteoblast proliferation by regulating RNAPII transcription activation.

Aims: Mechanical stimuli are crucial in regulating normal osteoblast function to maintain bone homeostasis. Decreased osteoblast proliferation is one of the main causes of osteoporosis induced by mechanical unloading. However, the underlying mechanism remains unclear. Cyclin-dependent kinase 12 (CDK12), a member of the CDK family, plays a key role in regulating cell proliferation. In this study, we aimed to investigate the role of CDK12 in osteoblast proliferation, and its function and underlying mechanism in decreased osteoblast proliferation caused by mechanical unloading. Methods: CDK12 small interfering RNA (siRNA) and CDK12 plasmid were used to knock down and overexpress CDK12 in osteoblasts, respectively. 5-ethynyl-2´-deoxyuridine (EdU) staining, cell counting, and flow cytometry were adopted to detect the effects of mechanical unloading on osteoblast cell proliferation and cell cycle. Immunofluorescence staining was performed to detect the cellular localization of CDK12. Real-time polymerase chain reaction (PCR) and western blot were performed to analyze the expression of CDK12 and downstream molecules. Results: Mechanical unloading significantly reduced the EdU-positive cell ratio and the total cell number of primary osteoblasts, accompanied by a marked decrease of S phase cell percentage. Meanwhile, the expression of CDK12, a key regulator for cell cycle and cell proliferation, was significantly downregulated by mechanical unloading. Loss and gain of function studies revealed that CDK12 knockdown inhibits osteoblast proliferation and S phase cell cycle progression, while CDK12 overexpression increases these parameters. Moreover, mechanical unloading altered the subcellular localization of CDK12, reduced the phosphorylation level of RNA polymerase II (RNAPII), and downregulated the expression of key DNA replication genes, including CDC6, CDT1, ORC2, and TOPBP1. Similar results were obtained in CDK12 knockdown osteoblasts. Furthermore, CDK12 overexpression rescued the decreased osteoblast proliferation and reduced S phase percentage induced by mechanical unloading. Conclusion: The present study demonstrates CDK12 as a critical regulator of osteoblast proliferation under mechanical unloading conditions. Mechanistically, mechanical unloading suppresses osteoblast proliferation by downregulating CDK12 expression, which results in decreased RNAPII phosphorylation and reduced expression of downstream core DNA replication genes. These findings provide a novel mechanistic insight into the inhibitory effects of mechanical unloading on osteoblast proliferation, and reveals CDK12 as a novel potential target for treating disuse osteoporosis.