Increased Bone Resorption during Lactation in Pycnodysostosis

Abstract

Pycnodysostosis, a rare autosomal recessive skeletal dysplasia, is caused by a deficiency of cathepsin K. Patients have impaired bone resorption in the presence of normal or increased numbers of multinucleated, but dysfunctional, osteoclasts. Cathepsin K degrades collagen type I and generates N-telopeptide (NTX) and the C-telopeptide (CTX) that can be quantified. Levels of these telopeptides are increased in lactating women and are associated with increased bone resorption. Nothing is known about the consequences of cathepsin K deficiency in lactating women. Here we present for the first time normalized blood and CTX measurements in a patient with pycnodysostosis, exclusively related to the lactation period. In vitro studies using osteoclasts derived from blood monocytes during lactation and after weaning further show consistent bone resorption before and after lactation. Increased expression of cathepsins L and S in osteoclasts derived from the lactating patient suggests that other proteinases could compensate for the lack of cathepsin K during the lactation period of pycnodysostosis patients.

Keywords: bone remodeling; cathepsin K; lactation; osteoclasts; osteocytes; pycnodystostosis.

Figure 1

Osteoclast cultures. Osteoclasts were generated from CD14⁺ cells with MCSF and RANKL for 4 weeks and subsequently fixed and stained for TRAcP (red); nuclei were visualized with DAPI (blue). Cells from both the healthy control (A) and the lactating patient (B) generated multinucleated osteoclasts. Control osteoclasts cultured on trabecular bone slices generated resorption trenches (C). This was in contrast to the osteoclasts of the patient where only small round resorption pits were visible (D). Confocal microscopy showed that actin rings, visualized with phalloidin-alexa488 (green), were abundantly present in cells of the control (E). These actin rings were hardly visible in cells of the patient (F). Staining the resorption area with alexa-647-labeled bisphosphonate (red) revealed less resorption by cells of the patient compared to the control (Figure 1E compared to Figure 1F). The nuclei were stained with DAPI (blue). TRAcP staining and resorption of osteoclasts from the patient during weaning is displayed in G,H. Scalebar 100 µm.

Figure 2

The total number of osteoclasts and resorption. (A) The number of osteoclasts was counted after 4 weeks of culture and the total number of osteoclasts was assessed. During the lactation period a lower number of osteoclasts was found in the PYCNO group compared to the control. * p < 0.05. (B) The from CD14⁺ cells generated osteoclasts were cultured on trabecular bone slices and resorption area was measured. The area resorbed by the PYCNO is significantly smaller compared to the control. * p < 0.05.

Figure 3

qPCR of cathepsins K, S, and L. Cathepsin K expression was very low in the patient (A). During lactation the expression of cathepsin L and S (B,C) was significantly elevated in the osteoclasts of the PYCNO patient. ** p < 0.01; *** p < 0.0001.