Defective co-activator recruitment in osteoclasts from microphthalmia-oak ridge mutant mice

Abstract

The three basic DNA-binding domain mutations of the microphthalmia-associated transcription factor (Mitf), Mitf(mi/mi), Mitf(or/or), and Mitf(wh/wh) affect osteoclast differentiation with variable penetrance while completely impairing melanocyte development. Mitf(or/or) mice exhibit osteopetrosis that improves with age and their osteoclasts form functional multinuclear osteoclasts, raising the question as to why the Mitf(or/or) mutation results in osteopetrosis. Here we show that Mitf(or/or) osteoclasts express normal levels of acid phosphatase 5 (Acp5) mRNA and significantly lower levels of Cathepsin K (Ctsk) mRNA during receptor activator of nuclear factor kappa B (NFkappaB) ligand (RANKL)-mediated differentiation. Studies using chromatin immunoprecipitation (ChIP) analysis indicate that low levels of Mitf(or/or) protein are recruited to the Ctsk promoter. However, enrichment of Mitf-transcriptional co-activators PU.1 and Brahma-related gene 1 (Brg1) are severely impaired at the Ctsk promoter of Mitf(or/or) osteoclast precursors, indicating that defective recruitment of co-activators by the mutant Mitf(or/or) results in impaired Ctsk expression in osteoclasts. Cathepsin K may thus represent a unique class of Mitf-regulated osteoclast-specific genes that are important for osteoclast function.

Figure 1. Mitf^or/or mice exhibit osteopetrosis that improves with age

A. Representative (see below) radiographic images of long bones (left in each panel) and digital microscopic images of Acp5 and hematoxylin stained 5 μM femur sections(right in each panel) from newborn (Left panel) and 30-day old (Right panel) WT, Mitf^wh/wh (mi-White), Mitf^or/or (mi-OR) and Mitf^mi/mi (mi/mi) mice are shown. Black arrow heads in newborn Mitf^or/or and Mitf^mi/mi radiographs indicate sclerotic lesions in mid-diaphysis and white arrowheads in 30-day old Mitf^or/or radiographs indicate sclerotic lesions in distal metaphysis of femurs. Black arrowheads in the Acp5 stained sections indicate osteoclasts positive for Acp5 enzyme activity. B-D. Bone and osteoclast parameters measured by histomorphometric analysis of Acp5 bone sections from new born and 30-day old WT, Mitf^wh/wh (mi-White), Mitf^or/or (mi-OR) and Mitf^mi/mi (mi/mi) mice are shown. B: BV/TV indicates the percentage of unresorbed trabecular bone volume to total bone surface; C:- N.OcS/BS indicates the number of osteoclasts per total bone surface and D:- Oc.S/BS indicates the percentage of osteoclast surface to total bone surface. For all three analyses, averages of n≥30 for newborn mice and n=20 for 30-day old mice in the respective genotypes is shown. * indicates statistical significance with p < 0.05, Student's t-test.

Figure 2. Mitf^or/or precursors form fewer functional multinuclear osteoclasts than WT in vitro

Precursors from either newborn (A) or 30-day old mice (B) of the indicated genotypes were cultured in the presence of CSF-1 and RANKL on gelatin coated wells for the formation of multinuclear osteoclasts (Ai and Bi) or on calcium phosphate coated wells for the formation of resorption pits (Aii and Bii). Representative digital microscopic images of Acp5 stained osteoclasts derived from newborn (Ai) or 30-day old (Bi) mice of the indicated genotypes. Figures Aii and Bii show representative digital microscopic images of resorption pits made by osteoclasts, from indicated genotype and age. Quantitation of the differentiation and functional assays is summarized in tables 1 and 2.

Figure 3. Mitf^or/or osteoclasts express normal levels of Acp5 mRNA and significantly lower levels of Cathepsin K

Average induction of Acp5 (A and D), Ctsk (B and E) and c-fms (C and F) mRNA levels in osteoclasts derived from newborn (left panels) or 30-day old (right panels) mice of indicated genotypes, at 24 or 72 hours in the presence of RANKL is shown. Average data (n=3), normalized to corresponding GAPDH and 18S mRNA levels, indicated as fold induction in the expression of each gene over CSF1 only treated (undifferentiated) controls, shown. Error bars indicate standard deviation and * indicates statistical significance (p<0.05, ANOVA).

Figure 4. Defective enrichment of PU.1 and Brg1 proteins at the Ctsk promoter in Mitf^or/or osteoclasts

Average enrichment (n=3) of Mitf (A), PU.1 (B) and Brg1 (C) proteins at the Ctsk promoter in WT, Mitf^wh/wh, Mitf^or/or and Mitf^mi/mi osteoclast precursors treated with CSF-1 alone or with CSF1 and RANKL for 72 h assayed by ChIP analysis. Error bars in all panels represent standard deviation and * indicates statistical significance (p<0.05, Student's t-test).