Aggrecan is required for chondrocyte differentiation in ATDC5 chondroprogenitor cells

Abstract

Aggrecan is an integral component of the extracellular matrix in cartilaginous tissues, including the growth plate. Heterozygous defects in the aggrecan gene have been identified as a cause of autosomal dominant short stature, bone age acceleration, and premature growth cessation. The mechanisms accounting for this phenotype remain unknown. We used ATDC5 cells, an established model of chondrogenesis, to evaluate the effects of aggrecan deficiency. ATDC5 aggrecan knockdown cell lines (AggKD) were generated using lentiviral shRNA transduction particles. Cells were stimulated with insulin/transferrin/selenium for up to 21 days to induce chondrogenesis. Control ATDC5 cells showed induction of Col2a1 starting at day 8 and induction of Col10a1 starting at day 12. AggKD cells had significantly reduced expression of Col2a1 and Col10a1 (p<0.0001) with only minimal increases in expression over time, indicating that chondrogenesis was markedly impaired. The induction of Col2a1 and Col10a1 was not rescued by culturing of AggKD cells in wells pre-conditioned with ATDC5 extracellular matrix or in co-culture with wild-type ATDC5 cells. We interpret our studies as indicating that aggrecan has an integral role in chondrogenesis that may be mediated through intracellular mechanisms.

Fig 1. Relative Aggrecan mRNA expression levels, as measured by qPCR, in lentiviral-transduced control cells and 5 AggKD cell lines confirm decreased Aggrecan expression in the AggKD cells.

Cells were cultured in media containing ITS for 12 days prior to being lysed for RNA preparation. Hatched bars represent unstimulated cells and solid bars represent cells stimulated with insulin to induce chondrogenesis. Data are shown as the mean ±1 SD for 4 biological replicates per group. ** p<0.01 versus control cells; ^## p<0.01 versus control cells +ITS.

Fig 2. Aggrecan knockdown ATDC5 chondroprogenitor cells have decreased Alcian blue staining and absorbance levels compared to control cells after ITS stimulation.

Cells were cultured in media containing ITS for 12 days at which they were fixed and stained with Alcian blue. (a) Representative stained cell culture plates are shown for each cell line. (b) Absorbance measurements after Alcian blue extraction are shown as the mean + 1 SD for 3 biological replicates per condition. Hatched bars represent unstimulated cells, and solid bars represent cells stimulated with ITS. *, p<0.01 versus control cells; ** p<0.01 versus control cells +ITS; †, p<0.05 versus control cells.

Fig 3. Chondrogenesis program is altered in Aggrecan knockdown ATDC5 chondroprogenitor cells.

mRNA expression levels of Col2a1 (a), Col10a1 (b), Sox9 (c) and Ihh (d) were determined by qPCR at day 0 through day 21 following chondrogenesis induction by the addition of ITS to culture media. Data for the control cells (solid line) and the two aggrecan knockdown cell lines, AggKD1 (dashed lines) and AggKD2 (dotted lines) are shown as the mean +1 SD for 4 biological replicates per condition. *, p<0.0001 versus AggKD lines.

Fig 4. Aggrecan knockdown ATDC5 cells are unresponsive to extracellular matrix cues that normally accelerate the chondrogenic program.

qPCR measurements to determine Col2a1 (a) and Col10a1 (b) expression were performed on RNA derived from lentiviral transduced control cells and 2 AggKD cell lines grown in cell culture plates that were untreated (hatched bars) or pre-conditioned with the extracellular matrix of control ATDC5 cells. qPCR measurements to determine Col2a1 (c) and Col10a1 (d) expression were performed on RNA derived from 2 AggKD cell lines grown individually (hatched bars) or co-cultured with wild-type ATDC5 cells. Before RNA extraction, wild-type ATDC5 cells were eliminated through puromycin selection. Data are shown as the mean +1 SD for 4 biological replicates per condition. *, p<0.05 versus corresponding unconditioned samples; **, p<0.01 versus corresponding unconditioned control samples.

Fig 5

(a) Phase contrast photomicrographs of ITS-stimulated ATDC5 cells on days 0, 12 and 21 in culture. Images were acquired at 10x magnification. Scale bar in the bottom right corner depicts 100 micrometers. (b) Hemocytometer counts of ITS-stimulated controls cells (solid line) and aggrecan knockdown cells AggKD1 (dashed line) and AggKD2 (dotted line) on days 0, 4 and 8. Data are shown as the mean +1 SD for 3 biological replicates per condition. *, p<0.0005 versus both aggrecan knockdown cell lines.

Fig 6. Proliferation is similar in control ATDC5 chondroprogenitor cells compared to aggrecan knockdown cells.

Cells cultured in the presence of ITS were assessed by flow cytometry with propidium iodine staining. Data, representing the sum of cells in the S and G2 phases of the cell cycle are shown as the mean + 1 SD for 3 biological replicates per condition. Control cells, solid line; AggKD 1, dashed line; AggKD 2, dotted line.