γ-Linolenic Acid Induces a Vitamin D Receptor-Independent Mineralization Program by Activating CaMKII–SMAD2/3 Pathway in Calvarial Osteoblasts

Vitamin D receptor (VDR) signaling is essential for osteoblast maturation and skeletal mineralization, yet the intracellular mechanisms linking VDR activity to matrix production remain poorly defined. Here, we show that vdr ⁻/⁻ calvarial osteoblasts initiate differentiation but fail to complete the transition to a mature, mineralizing state, exhibiting suppressed late-stage markers such as Dmp1 , Phex , and Col1a1 and defective nodule formation both in vivo and in vitro . Transcriptomic profiling revealed a SMAD network imbalance, with elevated inhibitory SMADs and attenuated phosphorylation of both SMAD1/5/9 and SMAD2/3. Whereas SMAD1/5/9 activation was preserved in vivo and restored by exogenous BMP2 in vitro , consistent with in vivo paracrine BMP availability, reduced SMAD2/3 phosphorylation persisted in both contexts, indicating a cell-autonomous defect. Mechanistically, vdr ⁻/⁻ osteoblasts displayed impaired intracellular Ca²⁺ dynamics and diminished CaMKII activation, with VDR/RXR occupancy detected near the Camk2g locus. Pharmacological CaMKII inhibition (KN-93) recapitulated the phenotype, reducing SMAD2/3 phosphorylation and mineralization and establishing CaMKII as an upstream regulator of SMAD2/3. A milk-based diet rescued skeletal defects in vdr ⁻/⁻ mice in a calcium-independent manner and selectively restored CaMKII–SMAD2/3 signaling. Metabolomic profiling identified the omega-6 fatty acid γ-linolenic acid (GLA) as an elevated circulating mediator; exogenous GLA restored Ca²⁺ flux, CaMKII activation, SMAD2/3 phosphorylation, matrix production, and mineralization, without reactivating Runx2 , Sp7 , or BMP–SMAD1/5/9 signaling, and dependent on CaMKII activity. These findings reveal a VDR-independent, GLA-inducible CaMKII–SMAD2/3 mineralization program, positioning metabolic modulation of calcium signaling as a strategy to restore osteoblast function under impaired vitamin D signaling.