γLinolenic Acid Induces a Vitamin D Receptor-Independent Mineralization Program by Activating CaMKII--SMAD2/3 Pathway in Calvarial Osteoblasts
- 1Molecular Genetics Laboratory, National Institute of Immunology, New Delhi, India
Abstract
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 y-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.
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BibTeX
@misc{biswas2026γlinolenic,
title = {{γLinolenic Acid Induces a Vitamin D Receptor-Independent Mineralization Program by Activating CaMKII--SMAD2/3 Pathway in Calvarial Osteoblasts}},
author = {T. Biswas and C. Chongtham and N. Kumari and Y. Saneja and N. K. Yadav and J. S. Maras and S. S. Kamat and G. A. Arimbasseri},
howpublished = {bioRxiv},
year = {2026},
month = jun,
doi = {10.64898/2026.06.10.731289},
url = {https://www.biorxiv.org/content/10.64898/2026.06.10.731289v2},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - γLinolenic Acid Induces a Vitamin D Receptor-Independent Mineralization Program by Activating CaMKII--SMAD2/3 Pathway in Calvarial Osteoblasts
AU - T. Biswas
AU - C. Chongtham
AU - N. Kumari
AU - Y. Saneja
AU - N. K. Yadav
AU - J. S. Maras
AU - S. S. Kamat
AU - G. A. Arimbasseri
PY - 2026
DA - 2026/06/10
PB - bioRxiv
DO - 10.64898/2026.06.10.731289
UR - https://www.biorxiv.org/content/10.64898/2026.06.10.731289v2
AB - 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 y-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.
ER -