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. 1998 Nov 10;95(23):13453-8.
doi: 10.1073/pnas.95.23.13453.

Impaired osteoclastic bone resorption leads to osteopetrosis in cathepsin-K-deficient mice

P Saftig  1 , E Hunziker, O Wehmeyer, S Jones, A Boyde, W Rommerskirch, J D Moritz, P Schu, K von Figura
Affiliations

Impaired osteoclastic bone resorption leads to osteopetrosis in cathepsin-K-deficient mice

P Saftig et al. Proc Natl Acad Sci U S A. .

Abstract

Cathepsin K is a recently identified lysosomal cysteine proteinase. It is abundant in osteoclasts, where it is believed to play a vital role in the resorption and remodeling of bone. Pycnodysostosis is a rare inherited osteochondrodysplasia that is caused by mutations of the cathepsin-K gene, characterized by osteosclerosis, short stature, and acroosteolysis of the distal phalanges. With a view to delineating the role of cathepsin K in bone resorption, we generated mice with a targeted disruption of this proteinase. Cathepsin-K-deficient mice survive and are fertile, but display an osteopetrotic phenotype with excessive trabeculation of the bone-marrow space. Cathepsin-K-deficient osteoclasts manifested a modified ultrastructural appearance: their resorptive surface was poorly defined with a broad demineralized matrix fringe containing undigested fine collagen fibrils; their ruffled borders lacked crystal-like inclusions, and they were devoid of collagen-fibril-containing cytoplasmic vacuoles. Assaying the resorptive activity of cathepsin-K-deficient osteoclasts in vitro revealed this function to be severely impaired, which supports the contention that cathepsin K is of major importance in bone remodeling.

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Figures

Figure 1
Figure 1
Targeted disruption of the cathepsin-K gene. (a) Strategy for inactivation of the cathepsin-K gene by homologous recombination in embryonic stem cells. (Part I) 10-kbp portion of the cathepsin-K gene, depicting salient structural information. Exons are indicated by open boxes, flanking introns by solid vertical lines. Horizontal bars, designated as cathepsin-K probes 3′ and 5′, denote the DNA probes used for Southern blot analyses. (Part II) Targeting vector pCK-Kpn(neo) with 5.3-kbp homology to the cathepsin-K gene locus. The neo-cassette was inserted into a HindIII restriction site in exon 7. Arrowhead indicates the direction of transcription of the neo gene. (Part III) Predicted ctsk gene locus after homologous recombination. (b) PCR analysis of tail-genomic DNA with an exon-specific PCR amplifying a 0.4-kb fragment in wild-type (+/+) mice, 0.4-kb and 1.6-kb fragment in heterozygous (+/−) mutants, and a 1.6-kb fragment in homozygous (−/−) cathepsin-K-deficient animals. (c) Northern blot analysis of cathepsin-K expression. Total RNA (10 μg) was hybridized by using murine cathepsin-K and glyceraldehyde-3-phosphate dehydrogenase cDNA probes. (d) RT-PCR of total bone RNA and cathepsin-K-specific primers. In +/+ mice, a 990-bp fragment is amplified, whereas in −/− mice there is no amplification. (e) Western blot analysis of cathepsin-K expression by using an antiserum against mouse cathepsin K (37). In +/+ bone extracts, the 46-kDa cathepsin-K precursor and the 30-kDa mature form are revealed. In −/− bones, no cathepsin-K gene product is apparent. Recombinant mouse cathepsin K served as a control.
Figure 2
Figure 2
Osteopetrosis in cathepsin-K deficient mice. (a) Radiographs of 16-week-old bones in cathepsin-K knockout (−/−) and control (+/+) mice. Left, femura. Center, humeri. Note extensive trabeculation of bone-marrow space (beginning at the distal end) in cathepsin-K-deficient mouse. Right, lumbar vertebrae (note the very dense and irregular distribution of spongiosa in the cathepsin-K-deficient mouse. (b and c) SEM images of 3-month-old proximal femura (b, with retained epiphyses) and tibiae (c, with epiphyses removed). Note the retention of cancellous bone in the shafts of cathepsin-K-deficient (−/−) bones. Field widths: b = 7.69 mm; c = 5.84 mm (d and e). Histological sections of the meta- and epiphysis of control (d) and cathepsin-K-deficient (e) femura embedded in methyl methacrylate and stained with McNeil’s tetrachrome. Note the unresorbed primary spongiosa in e. (f and g) Histological sections (sagittal plane) through the ventral half of thoracic vertebrae derived from control (f) and cathepsin-K-deficient (g) mice. BM, bone marrow; CB, cortical bone; GP, growth plate. [Bars = 400 μm (d–g).]
Figure 3
Figure 3
Ultrastructural changes in cathepsin-K-deficient tibial osteoclasts. (a and b) Control osteoclasts (OCL). At the resorption site (arrows), these cells protrude long microplicae comprising the characteristic ruffled border. The so-called “clear” zones are indicated by arrowheads. (c) Ruffled border of a control osteoclast depicting electron-dense inclusions (arrowheads). (d) Ruffled border of a cathepsin-K-deficient osteoblast, composed of numerous slender microplicae. Note the poorly defined resorption front. IS, interstitial tissue space; M, mineralized bone matrix. (e) Resorption interface between a cathepsin-K-deficient osteoclast and the mineralized bone matrix (M). Numerous fine collagen fibrils have been exposed by mineral removal by the osteoclast. (f and g) Resorption pits associated with cathepsin-K-deficient osteoclasts illustrating (at higher magnifications) the collagen fibrils that persist after digestion of the mineral substance (M). MV [in (g)], microvilli of osteoclast. (h) Well orientated and clearly exposed collagen fibrils in resorption pit associated with cathepsin-K-deficient osteoclast that are digested away at a straight resorption front (arrow). [Bars = 5 μm (a), 2 μm (b, c, d, h), 3 μm (e), 1 mm (f), and 0.5 μm (g).]
Figure 4
Figure 4
Cathepsin-K-deficient osteoclasts with impaired bone resorption. Fresh osteoclasts were isolated and seeded as a suspension onto dentine sections, and (a) the total number of resorption pits therein was then determined. Cathepsin-K-deficient (−/−) osteoclasts formed significantly fewer resorption pits than did control (+/+) or heterozygote (+/−) ones. (Two-sample t test for +/+ vs. −/−: P = 0.0001; for ± vs. −/−: P = 0.001). (b and d) Images of resorption pits produced in the confocal reflection light microscope (maximum brightness; ref. 29). On dentine slices seeded with control osteoclasts (b), the pits are deeper and more sharply defined than on those seeded with cathepsin-K-deficient (−/−) osteoclasts (d). (c and e) Topographic-map images of resorption pits. Each band of color (pink, superficial; blue, deep) represents 1 μm in the vertical direction. The control pit (c), with characteristically steep sides, has a maximum depth of approximately 13 μm, whereas one of similar area created by a cathepsin-K-deficient osteoclast (e) is only about 3 μm deep. Field widths of b–e are 143 μm (f) Resorption pits analyzed by 15-kV digital backscattered electron imaging, pseudocolor coded to scale the amount of residual matrix. In cathepsin-K −/− pits, a greater thickness (more colors) of unprocessed residual matrix was found. Field width of each part = 128 μm.
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