Inflammation-Mediated Age-Dependent Effects of Casein Kinase 2-Interacting Protein-1 on Osteogenesis in Mesenchymal Stem Cells
Mesenchymal stem cells (MSCs) are multipotent progenitors capable of differentiating into osteoblasts, adipocytes, and chondrocytes. Their differentiation potential is tightly regulated by transcription factors, signaling pathways, and environmental cues. Casein kinase 2-interacting protein-1 (CKIP-1), a negative regulator of osteogenesis, has been implicated in bone homeostasis through its interaction with Smurf1, an E3 ubiquitin ligase that degrades osteogenic signaling proteins. However, the age-dependent effects of CKIP-1 on MSC osteogenesis and its relationship with chronic inflammation remained poorly understood. This study investigated how CKIP-1 modulates MSC differentiation, bone mass, and inflammation-mediated osteogenic changes across different age groups.
CKIP-1 Deficiency Enhances Osteogenic and Adipogenic Differentiation in MSCs
MSCs isolated from CKIP-1 knockout (KO) and wild-type (WT) mice exhibited similar spindle-shaped morphology and surface marker profiles (CD44+, CD90+, CD106+, CD34−, CD45−), confirming their mesenchymal lineage. However, CKIP-1 KO MSCs demonstrated significantly enhanced osteogenic and adipogenic potential. After 7 days of osteogenic induction, alkaline phosphatase (ALP) activity in CKIP-1 KO MSCs was 1.5-fold higher than in WT cells (P < 0.05). By day 21, alizarin red staining revealed a 2.3-fold increase in mineralized nodule formation in CKIP-1 KO cultures. Quantitative PCR (qPCR) showed upregulation of osteogenic markers Runx2 (2.6-fold) and osteocalcin (2.1-fold) in KO MSCs (P < 0.05). Similarly, adipogenic induction for 6 days resulted in a 1.8-fold increase in oil red O-positive lipid droplets in CKIP-1 KO MSCs compared to WT cells (P < 0.05), though adipogenic marker PPAR-γ showed no significant difference. These findings highlight CKIP-1’s dual role in suppressing both osteogenesis and adipogenesis in MSCs.
Age-Dependent Effects of CKIP-1 on Bone Mass In Vivo
The impact of CKIP-1 on bone mass was age-dependent. Micro-CT analysis of 2-month-old (2M) mice showed no significant differences in trabecular bone volume/tissue volume (BV/TV) or bone mineral density (BMD) between WT and KO groups. In contrast, 18-month-old (18M) CKIP-1 KO mice exhibited a 25% increase in BV/TV (P = 0.02) and a 18% higher BMD (P < 0.05) compared to age-matched WT mice. Body weight differences also emerged with aging: 18M KO mice weighed 11 g more than WT controls, suggesting CKIP-1’s role in regulating age-related adiposity. These results indicate that CKIP-1’s inhibitory effects on bone formation become more pronounced with aging.
Inflammation Upregulates CKIP-1 Expression in Aging Bone Marrow
CKIP-1 expression in bone marrow was 4.3-fold higher in 18M WT mice compared to 2M WT mice at the mRNA level (P = 0.04), with a corresponding 3.1-fold increase in protein expression by western blot. However, in vitro-cultured MSCs from young and old mice showed no intrinsic age-related differences in CKIP-1 expression under normal or osteogenic conditions. This discrepancy suggested that the bone marrow microenvironment, rather than MSC aging itself, drives CKIP-1 upregulation.
To test the role of inflammation, MSCs were treated with interleukin-1β (IL-1β), a pro-inflammatory cytokine elevated in aged tissues. IL-1β (10 ng/mL) increased CKIP-1 mRNA by 3.2-fold (P = 0.03) and protein by 2.8-fold in vitro. In vivo, 2M WT mice injected with IL-1β (50 mg/kg, twice weekly for 12 weeks) showed a 3.2-fold increase in bone marrow CKIP-1 mRNA (P = 0.03) and a 30% reduction in trabecular BV/TV compared to PBS-treated controls (P < 0.05). These data establish inflammation as a critical mediator of CKIP-1 overexpression in aging bone marrow, leading to suppressed osteogenesis.
Mechanistic Insights into CKIP-1’s Regulatory Role
CKIP-1 inhibits osteogenesis by enhancing Smurf1-mediated degradation of osteogenic signaling proteins like Smad1/5 and Runx2. In adipogenesis, CKIP-1 interacts with histone deacetylase 1 (HDAC1) to repress C/EBPα transcription. The age-dependent increase in CKIP-1 expression correlates with chronic inflammation, which disrupts MSC differentiation balance toward adipogenesis at the expense of osteogenesis. This shift contributes to age-related bone loss and marrow adiposity.
Notably, while CKIP-1 deficiency enhanced both osteogenic and adipogenic differentiation in vitro, its in vivo effects were context-dependent. Young KO mice showed no bone mass differences, suggesting compensatory mechanisms or redundant pathways in early life. In contrast, aged KO mice exhibited higher bone mass, underscoring CKIP-1’s escalating inhibitory role in osteogenesis during aging.
Clinical Implications and Future Directions
Targeting CKIP-1 could offer therapeutic benefits for osteoporosis. Previous studies showed that CKIP-1 siRNA enhances bone formation in osteoporotic models. This study further suggests that anti-inflammatory therapies might mitigate age-related CKIP-1 upregulation, preserving MSC osteogenic capacity. However, the exact signaling pathways linking inflammation to CKIP-1 expression require further investigation. Potential mechanisms include NF-κB activation or epigenetic modifications induced by IL-1β.
Additionally, the dual role of CKIP-1 in osteogenesis and adipogenesis complicates therapeutic targeting. Selective inhibition of CKIP-1’s osteogenic suppression while preserving its adipogenic regulation may be necessary to avoid adverse metabolic effects.
Conclusion
CKIP-1 acts as a negative regulator of MSC osteogenesis, with effects magnified by age-related inflammation. In vivo, CKIP-1 expression in bone marrow increases with aging, driven by inflammatory cytokines like IL-1β, leading to reduced bone mass. These findings highlight the interplay between inflammation, aging, and MSC differentiation, positioning CKIP-1 as a potential target for treating age-related osteoporosis.
doi.org/10.1097/CM9.0000000000000951
Was this helpful?
0 / 0