Histone demethylase Kdm5c regulates osteogenesis and bone formation via PI3K/Akt/HIF1α and Wnt/β-catenin signaling pathways

Fractures have an extraordinarily negative impact on individuals’ quality of life and functional status. Nonunion or disability of fracture is a major health issue with important clinical, social, and economic implications. Mesenchymal stem cells (MSCs) play an indispensable role in the initiation of the fracture repair process including the formation of a callus which is replaced by new bone. The use of MSCs in the treatment of fractures is very attractive as they can reduce the time of healing and occurrence of nonunion. However, the effects of MSCs are often hindered by the harsh ischemic micro-environment at the fracture sites, such as low cell survival rate and differentiation in vivo. Histone modifications are one of the most important epigenetic regulations with the ability to control the fate of stem cells. Lysine demethylase 5C (Kdm5c) is frequently mutated in patients with X-linked intellectual disabilities, many of whom exhibit physical and behavioral abnormalities, including epilepsy, short stature, etc. In our previous study, we investigated the de-differentiated MSCs with enhanced osteogenic differentiation capacity and found that Kdm5c might be involved in regulating the properties of dedifferentiated osteogenic MSCs by PCR array. However, it is still unclear whether Kdm5c plays a role in osteogenesis, bone formation, and fracture repair. First, we established the Kdm5c conventional knockout mice. The MSCs were isolated to evaluate their osteogenic differentiation potential in vitro. The data showed that deletion of Kdm5c significantly impaired osteogenic differentiation as indicated by decreased mRNA levels of osteogenic markers, including alkaline phosphatase (Alp), Runt-related transcription factor 2 (Runx2), osteopontin (Opn) and osteocalcin (Ocn), as well as Alizarin Red S

Fractures have an extraordinarily negative impact on individuals' quality of life and functional status. Nonunion or disability of fracture is a major health issue with important clinical, social, and economic implications. 1 Mesenchymal stem cells (MSCs) play an indispensable role in the initiation of the fracture repair process including the formation of a callus which is replaced by new bone. The use of MSCs in the treatment of fractures is very attractive as they can reduce the time of healing and occurrence of nonunion. 2 However, the effects of MSCs are often hindered by the harsh ischemic micro-environment at the fracture sites, such as low cell survival rate and differentiation in vivo. 3 Histone modifications are one of the most important epigenetic regulations with the ability to control the fate of stem cells. Lysine demethylase 5C (Kdm5c) is frequently mutated in patients with X-linked intellectual disabilities, many of whom exhibit physical and behavioral abnormalities, including epilepsy, short stature, etc. 4 In our previous study, we investigated the de-differentiated MSCs with enhanced osteogenic differentiation capacity and found that Kdm5c might be involved in regulating the properties of dedifferentiated osteogenic MSCs by PCR array. 5 However, it is still unclear whether Kdm5c plays a role in osteogenesis, bone formation, and fracture repair.
First, we established the Kdm5c conventional knockout mice. The MSCs were isolated to evaluate their osteogenic differentiation potential in vitro. The data showed that deletion of Kdm5c significantly impaired osteogenic differentiation as indicated by decreased mRNA levels of osteogenic markers, including alkaline phosphatase (Alp), Runt-related transcription factor 2 (Runx2), osteopontin (Opn) and osteocalcin (Ocn), as well as Alizarin Red S staining ( Fig. S1). At 5 days and 2 months of age, it was observed that Kdm5c knockout (KO) mice had a smaller body size compared to wild-type (WT) littermates (Fig. S2). The whole skeleton of Kdm5c KO mice was smaller than that of WT littermates (Fig. 1A). The lengths of the rib, spine (1st thoracic to 1st lumber spine), humerus, ulna, radius, femur, and tibia were shorter in Kdm5c KO mice, as compared with that of WT littermates (Fig. 1BeF). The microCT analysis showed Kdm5c KO mice exhibited decreased trabecular bone mineral density, bone volume (BV), bone volume fraction (BV/TV), and trabecular number at 8 weeks of age (Fig. 1G, H). Simultaneously, the mineral apposition rate and bone formation rate of both groups were assessed in non-decalcified histological sections of femurs by Calcein-Alizarin Red labeling assay. The endocortical bone formation rate, median mineralizing surface per unit of the bone surface, and endocortical mineral apposition rate were reduced in the Kdm5c KO mice compared to WT littermates (Fig. S3A, B), suggesting a decreased endocortical bone formation. The immunohistochemistry (IHC) staining showed that OPG, OCN, and OPN were decreased in the femur's trabecular bone and bone cortex in Kdm5c KO mice (Fig. S3C, D).
Next, we isolated the total RNA of bone marrow MSCs from Kdm5c KO and WT mice and performed RNA sequencing (RNAseq) analysis. Among the 724 differentially expressed genes, 112 genes were up-regulated and 612 genes were down-regulated (Fig. S4A). The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed Kdm5c KO MSCs were enriched with a variety of signaling pathways closely related to bone development, such as PI3K-Akt, Wnt, Hippo, and Transforming growth factor-b (TGFb) signaling pathways, etc (Fig. 1I). Downregulation of genes associated with PI3K/Akt and Wnt signaling pathways were shown in Figure S4B. The Western blot analysis also showed the levels of p-Akt, b-catenin, and HIF1a were all decreased in MSCs isolated from Kdm5c KO mice (Fig. 1J). In addition, the IHC staining of the tibia revealed significantly reduced p-Akt, b-catenin, and HIF-1a in 2-month-old Kdm5c KO mice compared with WT littermates (Fig. S4C).
Then, mouse MSCs were transduced with lentivirus carrying Kdm5c or empty vector. Stable cell lines of MSCs (Kdm5c-MSC and Vector-MSC) were established. After 5 days of osteogenic induction, the mRNA expression levels of Alp, Opn, Bmp2, Runx2, and Ocn were significantly increased in Kdm5c-MSC (Fig. S5A). The Alizarin Red S staining showed that the mineralization of MSCs was also increased by Kdm5c over-expression at 14 days after osteogenic induction (Fig. 1K). The levels of p-Akt, b-catenin, and HIF1a were all increased by Kdm5c over-expression (Fig. S5B).
Finally, we established the mouse open femur fracture model, and Kdm5c-MSC and Vector-MSC were locally injected into the fracture sites. The Micro-CT result revealed that mice treated with Kdm5c-MSC transplantation displayed better fracture healing, significantly higher bone mineral density and bone volume fraction, and better mechanical properties (Fig. 1LeN). Increased levels of OCN and HIF1a in the callus tissue of Kdm5c-MSC transplantation mice could be observed (Fig. 1O).
Taken together, our data showed that Kdm5c deletion mice displayed decreased bone mass and abnormal bone development, with multiple signaling pathways inhibited, especially the PI3K/Akt/HIF1a and Wnt/b-catenin pathways (summarized in Fig. S6). Kdm5c-modified MSCs could be used for accelerating fracture healing, which may have clinical implications for the delayed unions.

Ethics declaration
All procedures were performed in strict accordance with the guidelines of the Animal Welfare and Ethics Committee of Guangzhou University of Chinese Medicine (No. 20190617017).