Bone resorption induced by parathyroid hormone is strikingly diminished in collagenase-resistant mutant mice (original) (raw)

Mice. To obtain sufficient r/r mice for the experiments to be described, we chose to use the progeny of r/r breeding pairs of the Col1a1tmlJae mice. Homozygous offspring were genotyped using a PCR-based method (see below) rather than by the more tedious method of Southern analysis reported previously (30). Wild-type (+/+) controls comprised C57BL/6, 129 strain, from which the J1 embryonic stem cells were derived, as well as the +/+ progeny of r/+ (heterozygous) breeding pairs.

Genotyping. We developed a rapid, nested PCR method with genomic DNA as template for genotyping. The DNA Thermal Cycler from Perkin-Elmer Corp. (Norwalk, Connecticut, USA) was used. We took advantage of changes in DNA sequence in the mutant IV construct (29) used for gene targeting in the Col1a1tmlJae mice. In the mutant construct, a downstream _Kpn_I site (within an intron) was destroyed but the _Kpn_I site in exon 40, where the amino acids surrounding the collagenase cleavage site are encoded in Col1a1, was retained and a new _Sph_I site was introduced in exon 40. First-round PCR was carried out using 1 μg of genomic DNA, a forward oligonucleotide primer in intron 35 (5′-TGAGACACGAGGCATGGGACC-3′) and a reverse primer in intron 43 (5′-GCATGTCTGAAGAAGAGGTCT-3′) to obtain sufficient amplified DNA. Second-round PCR was carried out using the first-round product as template with a forward oligonucleotide primer in intron 38 (5′-GTGAGTATCTGTGGTTCTGGA-3′) and a reverse primer in intron 41 (5′-CAGGGGGACTGGCTAGGAGGT-3′). Conditions were as follows: genomic DNA (∼1 μg/ml), 1.0 μl, as template for the first round; primers (50 mM), 0.5 μl of each; 5× PCR buffer (Tris-HCl [pH 8.5, 300 mM] and [NH2]SO4, [75 mM]), 10.0 μl; MgCl2 (25 mM), 4.0 μl; dNTP mixture (2.5 mM), 5.0 μl; sterile water to a total volume of 50 μl. Template for the second round was 1 μl of first-round product. The above samples were denatured for 2 min at 97°C. After reducing block temperature to 90°C, 1 μl of Taq polymerase (5 U/μl; Fisher Scientific, Pittsburgh, Pennsylvania, USA) was added to achieve a “hot start.” Temperatures were then 94°C for 30 s, 62°C for 40 s, 72°C for 2 min for 35 cycles, followed by a final extension time of 5 min at 72°C. Restriction enzymes were obtained from Promega Corp. (Madison, Wisconsin, USA). For restriction enzyme (Promega Corp.) digestion, 6.0 μl of the second-round PCR product was incubated with 0.5 μl of either _Sph_I or _Kpn_I at 10 U/μl, with 2.0 μl of the appropriate 10× buffer in a total volume of 20.0 μl. The predicted size, in bp, of the digestion products is: _Sph_I +/+, 986; _Sph_I r/r, 562,428; _Kpn_I +/+, 409, 360, 217; _Kpn_I r/r, 581, 409. Whenever sufficient genomic DNA was amplified, +/+, r/+, and r/r animals could readily be distinguished (data not shown).

Effects of PTH on bone resorption in vivo. To measure responses of calvarial bone to PTH, we followed the procedures developed by Boyce and coworkers (31, 32). Synthetic human PTH(1–34) was obtained from A. Khatri (Endocrine Unit, Massachusetts General Hospital, Boston, Massachusetts, USA) and was dissolved in vehicle (1 mM HCl, 0.1% BSA). PTH, at 10 μg in 25 μl or 2 μg in 10 μl, or vehicle in the same volume, were injected subcutaneously four times daily for 3 days, using a Hamilton syringe, into the subcutaneous tissue overlying the right side of the calvariae. After sacrifice by CO2 narcosis, calvariae were removed intact, soft tissues gently dissected, and the calvariae were fixed in 10% phosphate-buffered formalin for 24 h for further processing and analysis.

Tissue processing and analysis. After fixation, calvariae were decalcified in 14% EDTA for 7–8 days and then dehydrated in graded alcohol. Calvariae were then bisected perpendicular to the sagittal suture through the central portion of the parietal bone parallel to the lambdoidal and coronal sutures and paraffin embedded. Four to six 5-μm-thick representative, nonconsecutive step sections were cut. The sections were routinely stained with hematoxylin and eosin (H&E). The noninjected side was shortened after dissection to distinguish the two sides. To facilitate histomorphometric measurements, a standard length of 5 = mm of each section from the edge of the sagittal suture to the muscle insertion at the lateral border of each bone was used.

Small cavities filled with bone marrow are present within the calvariae of normal mice, and most of these are adjacent to sutures in the midline, anterior, posterior, and lateral aspects of the calvariae. After treatment with PTH and other resorption-stimulating agents, osteoclasts enlarge these cavities and extend them towards the middle portion of the parietal bones. In untreated mice, the middle of the parietal bone is typically composed of solid bone matrix, with few, if any marrow cavities. Thus, the resorptive activity after PTH treatment can be assessed quantitatively by measuring the area of these bone marrow spaces at multiple sites in the calvarial bones and comparing mean values with those in controls. The total areas of bone and marrow cavities (i.e., bone and marrow between the inner and outer periosteal surfaces within the 5 mm length from the sagittal suture) were measured on digitized images using the National Institutes of Health (NIH; Bethesda, Maryland, USA) Image program for the Macintosh computer (Apple Computer Inc., Cupertino, California, USA). The amount of resorptive activity was estimated by expressing the bone marrow area as a percentage of the total bone area to accommodate variations in the absolute amount of bone resorption among animals (total resorption area % = Σ areas of bone marrow cavity / Σ total bone areas × 100). Active bone resorption was also estimated by measuring the number of osteoclasts along the interface between bone and bone marrow, expressed as a percentage of the total length of this interface (active resorption surface % = Σ rough crenated surface with associated osteoclasts / total length of interface between bone and marrow cavity × 100). In some samples, the number of osteoclasts was counted within the marrow cavity and expressed as number/mm2 of the total bone area within the 5 = mm standard length). Osteoclasts were identified by their morphology and location in scalloped lacunae, and their presence was confirmed by staining for tartrate-resistant acid phosphatase (TRAP) (33).

To reduce measurement errors, at least two of the most central sections among all sections from each sample were examined and quantified. Each area of bone resorption was digitized twice using the NIH Image program, and two readings were averaged. All measured areas of bone resorption within each section were then summed. Statistical significance was determined using ANOVA.

Calcemic responses to PTH. To measure calcemic responses, synthetic human PTH(1–34) (15 μg) or vehicle was injected once intraperitoneally in groups of +/+ and r/r mice of different ages, and blood ionized calcium concentration, [Ca2+], was determined with the 634 ISC Ca2+/pH Analyzer from Ciba-Corning Diagnostics Corp. (Medfield, Massachusetts, USA) at intervals from 0.5 up to 10 h after injection. Statistical significance was determined using Student's t test.

In situ hybridization. Paraffin sections were used. Osteoclasts were further identified by in situ hybridization using a [35S]α-UTP 5′ antisense riboprobe composed of sequences in the 3′ untranslated region of the mouse 92-kDa gelatinase cDNA (18), a gift from K. Tryggvason (Karolinska Institute, Stockholm, Sweden). For the mouse collagenase, we prepared an antisense [35S]α-UTP 5′ riboprobe composed of the _Nco_I/_Avr_II sequences that cover the open reading frame through the zinc-binding site in the cDNA (16), a gift from Y. Eeckhout (University of Louvain Medical School, Brussels, Belgium). Sense riboprobes were used as controls.