Understanding cellular biological impacts of SATB2 mutations in osteoblasts
OA Version
Citation
Abstract
BACKGROUND: SATB2 Associated Syndrome (SAS) is a disorder which impacts neurological and osteogenic development, leading to a wide variety of clinical phenotypes and behaviors in patients. It is currently understood that SAS develops from altered expression of the SATB2 locus. SATB2 is a higher order transcription factor, with the ability to organize chromatin to regulate the expression of many cell-type specific differentiation programs. The impact of SATB2 at a molecular level is not well understood and requires more research to better understand and treat patients with SAS.
OBJECTIVE: There are two objectives to this research. One objective is to determine if activation or inhibition of protein kinase C (PKC) impacts the ability of SATB2 to bind DNA. The other objective is to determine if nuclear structure is affected by SATB2 mutations.
METHODS: The experiments in this study utilized mouse osteoblast (MC3T3) cells. In one set of experiments, cells were treated with either an activator, PMA, or inhibitor, Cal C, of PKC. Immunostaining for SATB2 was carried out to quantify the amount of SATB2 in the nucleus after treatment. Once treated, MC3T3 cells were imaged using a confocal microscope to quantify nuclear fluorescence of SATB2. To assess nuclear structure, lamin B1 staining was used. Wild type and SATB2 mutant cells were immunostained for lamin B1. These cells were imaged using a confocal microscope to examine nuclear structure.
RESULTS: The impact PKC has on SATB2 is unclear. It was expected that activation of PKC by PMA would decrease fluorescence while PKC inhibition by Cal C would increase fluorescence, these expectations were not strictly met. PMA treated cells demonstrated variable nuclear fluorescence, demonstrating expected results only in 2 out of 3 studies. Cal C treated cells did not increase fluorescence in any of the 3 experiments, instead fluorescence values were comparable to control values.
Nuclear structure was significantly impacted by SATB2 mutations. Mutated, nonfunctional SATB2 C8 cells, demonstrated an increase in nuclear aberrations and varied cell structure. Cell shape was greatly impacted by mutations. WT cells demonstrated uniform appearance with apparent nuclear infoldings throughout experiments. C8 cells varied in nuclear structure and size, with limited nuclear infoldings. WT cells also expressed lamin throughout the cell, with the most intense fluorescence seen in infoldings and surrounding the cell. C8 cells typically expressed the highest lamin fluorescence around the wall of the nucleus.
CONCLUSIONS: More research is necessary to understand the impact PKC has on SATB2. There is a trend toward decreased and localized nuclear SATB2 function in cells inhibited by PKC by PMA, but poor understanding of the role of Cal C on SATB2 function. Nuclear structure is clearly impacted by SATB2 dysregulation. Cells demonstrate great phenotypical variability when SATB2 is nonfunctional, including increased mutations and decreased nuclear infoldings.
Description
2025