Lamin A/C-Dependent Translocation of Megakaryoblastic Leukemia-1 and β-Catenin in Cyclic Strain-Induced Osteogenesis

Lamins are intermediate filaments that play a crucial role in sensing mechanical strain in the nucleus of cells. β-catenin and megakaryoblastic leukemia-1 (MKL1) are critical signaling molecules that need to be translocated to the nucleus for their transcription in response to mechanical strain that induces osteogenesis. However, the exact molecular mechanism behind the translocation of these molecules has not been fully investigated. This study used 10% cyclic strain to induce osteogenesis in the murine osteoblast precursor cell line (MC3T3). The translocation of β-catenin and MKL1 was studied by performing knockdown and overexpression of lamin A/C (LMNA). Cyclic strain increased the expression of osteogenic markers such as alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2), and enhanced ALP staining after seven days of incubation. Resultantly, MKL1 and β-catenin were translocated in the nucleus from the cytoplasm during the stress-induced osteogenic process. Knockdown of LMNA decreased the accumulation of MKL1 and β-catenin in the nucleus, whereas overexpression of LMNA increased the translocation of these molecules. In conclusion, our study indicates that both MKL1 and β-catenin molecules are dependent on the expression of LMNA during strain-induced osteogenesis.

Extrahepatic 25-Hydroxylation of Vitamin D3 in an Engineered Osteoblast Precursor Cell Line Exploring the Influence on Cellular Proliferation and Matrix Maturation during Bone Development

Osteoblastic precursors experience distinct stages during differentiation and bone development, which include proliferation, extracellular matrix (ECM) maturation, and ECM mineralization. It is well known that vitamin D plays a large role in the regulation of bone mineralization and homeostasis via the endocrine system. The activation of vitamin D requires two sequential hydroxylation steps, first in the kidney and then in the liver, in order to carry out its role in calcium homeostasis. Recent research has demonstrated that human-derived mesenchymal stem cells (MSCs) and osteoblasts can metabolize the immediate vitamin D precursor 25-dihydroxyvitamin D3 (25OHD3) to the active steroid 1α,25-dihydroxyvitamin D3 (1,25OH2D3) and elicit an osteogenic response. However, reports of extrahepatic metabolism of vitamin D3, the parental vitamin D precursor, have been limited. In this study, we investigated whether osteoblast precursors have the capacity to convert vitamin D3 to 1,25OH2D3 and examined the potential of vitamin D3 to induce 1,25OH2D3 associated biological activities in osteoblast precursors. It was demonstrated that the engineered osteoblast precursor derived from human marrow (OPC1) is capable of metabolizing vitamin D3 to 1,25OH2D3 in a dose-dependent manner. It was also demonstrated that administration of vitamin D3 leads to the increase in alkaline phosphatase (ALP) activity associated with osteoblast ECM maturation and calcium deposits and a decrease in cellular proliferation in both osteoblast precursor cell lines OPC1 and MC3T3-E1. These findings provide a two-dimensional culture foundation for future three-dimensional engineered tissue studies using the OPC1 cell line.