Severe Extracellular Matrix Abnormalities and Chondrodysplasia in Mice Lacking Collagen Prolyl 4-Hydroxylase Isoenzyme II in Combination with a Reduced Amount of Isoenzyme I^*

Generation of Mouse Lines with Inactivated P4ha1 and P4ha2 Genes

Establishment of the knock-out mouse line for C-P4H-I by insertion of a lacZ-PGK-neo cassette into exon 2 of the P4ha1 gene (Fig. 1A) has been described previously (12). To generate a knock-out construct for the inactivation of C-P4H-II, the SvJ mouse cosmid library (Stratagene) was screened with a mouse α(II) subunit cDNA (6) as a probe, and BAC mouse library screening services (Genome Systems) were used to obtain 5′ genomic clones for the P4ha2 gene (13). The genomic clones obtained were used to build the P4ha2 targeting construct that contained 1.2- and 4-kb genomic arms and the 4-kb lacZ-PKG-neo cassette inserted in-frame into the third exon of the P4ha2 gene after the translation initiation codon (Fig. 1B). The NotI linearized targeting construct was electroporated into R1 embryonic stem cells (kindly provided by Dr. A. Nagy, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Canada) and selected with G418, and genomic DNA isolated from G418-resistant colonies was screened by PCR. Genomic insertion of the targeting construct was screened using a forward primer from intron 2 of the P4ha2 gene and a reverse primer from the 5′ end of the LacZ gene (Fig. 1B and Table 1). The wild-type allele was identified using the same forward primer and a reverse primer from exon 3 of the P4ha2 gene (Fig. 1B and Table 1). Correct targeting was confirmed by Southern blotting analysis of EcoRI-digested genomic DNA with a 450-bp probe from intron 2 of the P4ha2 gene (Fig. 1B). Correctly targeted ES cell clones were injected into C57BL blastocysts, and two separate mouse lines were generated by standard methods and backcrossed with C57BL mice. A forward primer from intron 2 and a reverse primer from intron 3, amplifying a 600-bp fragment, were used for genotyping the wild-type P4ha2 allele, and a primer pair from the LacZ gene, amplifying a 850-bp fragment, was used for the targeted P4ha2 allele (Fig. 1B and Table 1).

To generate P4ha1^+/−;P4ha2^−/− mice, P4ha1^+/− and P4ha2^−/− mice were first cross-bred to obtain double heterozygous P4ha1^+/−;P4ha2^+/− mice, which were then intercrossed with the P4ha2^−/− mice. The genotypes of the offspring were analyzed by PCR assay using a forward primer from intron 1 of the P4ha1 gene together with a reverse primer either from exon 2 or from the LacZ gene, producing 1.5-kb and 850-bp fragments corresponding to wild-type and mutant alleles, respectively (Fig. 1A and Table 1). For the P4ha2 gene, a forward primer from intron 2 was used with a reverse primer from intron 3 or from the LacZ gene, producing a 1.9-kb fragment from the wild-type and a 2.0-kb fragment from the mutant allele, respectively (Fig. 1B and Table 1).

The analyses described in this study were carried out with mice that had been backcrossed at least 10 times. Growth of the gene-modified mice was monitored by measuring the body weights of the mutant mice and their littermates at P0 and then weekly between 5 and 20 weeks.

The animal maintenance and handling was performed according to a license approved by the Animal Ethical Committee and Animal Care and Use Committee of the University of Oulu. The animal experiments were performed according to a protocol approved by the Provincial State Office of Southern Finland.

Isolation and Culture of Primary Chondrocytes

For chondrocyte isolation, growth plates from the long bones of newborn mice were dissected and placed in Hanks' buffered salt solution (Gibco) followed by digestion with 0.25% trypsin-EDTA (Life Technologies) for 30 min at 37 °C and 195 units/ml collagenase (Worthington Biochemical Corporation) in Hanks' buffered salt solution for 2 h. Subsequently, the chondrocytes were plated at a density of 4 × 10⁵ cells/well in 6-well plates and grown in monolayer cultures in high glucose DMEM (Gibco) supplemented with 5–10% fetal bovine serum (Hyclone), 100 units/ml penicillin, 0.1 μg/ml streptomycin (BioWhittaker), and 50 μg/ml ascorbic acid (Wako). Depending on the experiment, the cells were cultured either in 21% oxygen (normoxia) or exposed to 1% O₂ (hypoxia) balanced with 5% CO₂ and 95% N₂ for 24 h in an InVivo2 400 hypoxia work station (Ruskinn Technologies). In all experiments, the medium was changed every second day.

Isolation of RNA and Quantitative Real Time RT-PCR

Newborn mouse growth plates were dissected immediately after the mice had been sacrificed, snap frozen in liquid nitrogen, and stored at −70 °C. Total RNAs from the growth plates or from primary mouse chondrocytes were isolated using the E.Z.N.A. Total RNA kit (Omega Bio-Tek). The concentration of the RNA samples was measured by UV spectroscopy. The iScript cDNA synthesis kit (Bio-Rad) was used for reverse transcription according to the manufacturer's instructions. The primers used for quantitative real time RT-PCR (qPCR) analysis of P4ha1, P4ha2, P4ha3, Col2a1, Col9a1, Aggrecan, Sox9, Bip, and Chop mRNAs are given in Table 2. The reactions were carried out with iTaq SYBRGreen Supermix with ROX (Bio-Rad) and a Stratagene MX3005 thermocycler, and the mRNA expression levels were calculated relative to β-actin.

C-P4H Activity Assay

Chondrocytes for the C-P4H activity assay were lysed on ice in a buffer containing 137 mm NaCl, 20 mm Tris-HCl, pH 8, 10% glycerol, 1% Nonidet P-40, and Complete proteinase inhibitor without EDTA (Roche). Bio-Rad protein assay (Bio-Rad) was performed for protein determination. C-P4H activity was assayed by a method based on measurement of the formation of 4-hydroxy [¹⁴C]proline in a [¹⁴C]proline-labeled protocollagen substrate consisting of nonhydroxylated pro-α chains of chick type I procollagen (15).

Skeletal Preparation, Histological, in Situ Hybridization, and Microcomputed Tomography (μCT) Analyses

To visualize the whole skeleton, deskinned and eviscerated newborn mice were stained with alizarin red (for bone) and Alcian blue (for cartilage) using standard procedures (16). For histological analyses, limbs dissected from E13.5, E17.5, newborn (P0), and P21 mice were fixed in freshly prepared 4% paraformaldehyde in PBS at 4 °C. Specimens were processed for paraffin embedding, sectioning, and staining with hematoxylin and eosin according to standard histological procedures (17). Sections from newborn proximal tibias were stained with Masson's trichrome and picrosirius red staining kit (Abcam) to visualize collagen. Olympus BX51 microscope equipped with polarizing filters was used for birefringence. In situ hybridization using [³⁵S]UTP-labeled riboprobes for Col2a1, Col10a1, and Spp1 was conducted on tissue sections as previously described (17).

Femoral and tibial lengths of newborn and 12-week-old female mice were determined by scanning hind limbs fixed in 10% neutral buffered formalin using a Skyscan 1176 μCT with high resolution settings (9 μm isotropic voxel size). To assess the degree of kyphosis, full body scans were performed using 35-μm isotropic voxel size. The kyphotic index, a measure of spine deformity, was calculated as described previously (18). Briefly, kyphotic index = AB/CD, where AB is the distance between the bodies of the first sacral and first thoracic vertebrae, and CD is the greatest distance from the line AB to the vertebral body.

BrdU Labeling and TUNEL Staining

To assess chondrocyte proliferation in embryos at E15.5, pregnant females were injected intraperitoneally with 100 μg of BrdU and 12 μg of fluorodeoxyuridine/g of body weight 2 h prior to sacrifice. The embryos were delivered by caesarian section, and their hind limbs were dissected, fixed, and embedded into paraffin. Proliferation analysis in the proximal tibial growth plates of 3-week-old females was done by injecting BrdU and fluorodeoxyuridine as above 6 h before tissue harvesting. Longitudinal sections across the tibia and femur were prepared. To detect actively proliferating cells, the nuclei that had incorporated BrdU were identified using a BrdU staining kit (Invitrogen). For terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay, paraffin sections from the hind limbs of newborn mice were permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate. The TUNEL assay was subsequently carried out using an in situ cell death detection kit (Roche) according to the manufacturer's instructions.

Amino Acid and Thermal Stability Analyses of Collagen

Lyophilized mouse tibial bone and fresh growth plates from newborn mice were transferred to prepyrolyzed hydrolysis tubes (6 × 60-mm; Duram), which were then inserted into a 50-ml hydrolysis vial containing 1 ml of 5.7 m hydrochloric acid solution (Sigma-Aldrich). Each sample tube and the hydrolysis vial were purged with nitrogen to remove the air (oxygen), and vapor-phase hydrolysis was then carried out at 165 °C for 50 min. The tubes containing the hydrolyzed samples were removed to a desiccator connected to a vacuum pump, and the samples were dried overnight. The hydrolysate from each tube was dissolved in 50 μl of 20 mm HCl and then filtered with an Ultrafree-MC (0.45 μm; Millipore). Amino acid derivatization was performed with a Waters AccQ·Tag chemistry package according to the manufacturer's instructions. The derivatized amino acids were analyzed with a Shimadzu Prominence HPLC with its fluorescence detector set at 250/395 nm.

The thermal stability of the pepsin-resistant fibril-forming collagens was analyzed from newborn mouse growth plates. Protein was extracted from the growth plates in 4 m GuHCl using TissueLyser LT (Qiagen) with 5-mm steel beads. The remaining pellet was washed with H₂O and digested with pepsin in 0.5 m acetic acid for 48 h. After neutralization, soluble collagens were subjected to digestion with a mixture of trypsin and chymotrypsin for 2 min at various temperatures (19). The samples were analyzed by 8% SDS-PAGE followed by Coomassie Blue staining.

Atomic Force Microscopy

For atomic force microscopy (AFM) analysis, tibiae were harvested from newborn animals and snap frozen in liquid nitrogen, and 30 μm sections were prepared using a cryostat. Cartilage slices were collected on microscope slides for AFM imaging and mechanical testing of the proximal tibial growth plate. AFM measurements were carried out using a NanoWizard AFM (JPK Instruments) in combination with an inverse Axiovert 200 optical microscope (Carl Zeiss MicroImaging). For reduced influence of ambient noise, the optical microscope was placed on a Micro 60 active isolation table (Halcyonics), and the whole setup was placed into a 1-m³ soundproof box. To precisely position the cantilever within the proliferative zone of mouse samples, the microscope was equipped with a CCD camera (The Imaging Source Europe). The AFM had a maximum lateral scan range of 100 × 100 μm² and a vertical range of 15 μm. For contact mode imaging and indentation measurements, silicon nitride MLCT microcantilevers with pyramidal tip (Bruker) and a nominal spring constant of 10 mN/m were used. The force constant of all cantilevers was determined individually using the thermal noise method (20). All images were recorded in air with a resolution of 512 × 512 pixels² at a line rate of 1 Hz. After recording overview images of 100 × 100 μm², the scan size was reduced to record high resolution images in the area of interest. All indentation experiments were carried out in phosphate-buffered saline, pH 7.4. On an area of 3 × 3 μm², 25 × 25 force indentation curves were recorded at 1 Hz. Two independent animals were analyzed of each genotype (each at 25 × 25 different positions). The Young's modulus was determined at each indentation position by fitting a modified Hertz model (pyramidal indenter) to the respective approach curve, using the JPK data processing software (version 4.2.20; JPK Instruments AG),

where F is the force which is required to push the tip into the sample, δ is the indentation depth, E is the Young's modulus, ν is the Poisson ratio which was assumed to be 0.5, and α represents the tip half-opening angle (17.5° for the cantilevers used) (21). The contact point was determined manually for each force curve, and the fit range was limited to a maximum indentation depth of 500 nm. For generating histograms and fitting Gaussian distributions to the histograms, the data analysis software Origin 8.0 (OriginLab Corporation) was used.

Transmission Electron Microscopy

Proximal tibial epiphyses from newborn control and P4ha gene-modified mice were microdissected and fixed in a 1% glutaraldehyde and 4% formaldehyde in 0.1 m phosphate buffer. The samples were postfixed in 1% osmium tetroxide, dehydrated in acetone, and embedded in Epon LX 112 (Ladd Research Industries). Thin sections (80 nm) were cut with a Leica Ultracut UCT ultramicrotome and stained with uranyl acetate and lead citrate. The samples were examined in a Philips CM100 TEM with a Morada CCD camera (Olympos Soft Imaging Solutions GMBH) for image capture or in a Tecnai G2 Spirit with Veleta and Quemesa CCD cameras (FEI Company).

Statistical Analyses

The statistical analyses were performed using Student's two-tailed t test. The area under the curve of the growth curves was calculated using the method of summary measures (22, 23). The data are shown as the means ± S.D. Values of p < 0.05 were considered statistically significant (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

Generation of C-P4H-II null (P4ha2^−/−) Mice and Mice in Which Homozygous Inactivation of C-P4H-II Is Combined with Heterozygous Inactivation of C-P4H-I (P4ha1^+/−;P4ha2^−/−)

We have previously shown that homozygous targeted disruption of the P4ha1 gene coding for the C-P4H-I α(I) subunit (Fig. 1A) leads to embryonic death between E10.5 and E11.5, whereas heterozygous P4ha1^+/− mice are born in the expected Mendelian ratios, are fertile, have a normal life span, and display no obvious phenotypic abnormalities (12). To study the in vivo roles of C-P4H-II, we established a mouse line lacking C-P4H-II activity by disrupting the P4ha2 gene through insertion of a lacZ-PGK-neo cassette into exon 3 in-frame with the translation initiation codon (Fig. 1B). Correctly targeted ES cells were injected into mouse blastocysts, and heterozygous mice were generated by routine methods. Cross-breeding of the P4ha2^+/− mice produced all three genotypes in the expected Mendelian ratio (data not shown). The homozygous P4ha2^−/− mice were viable and fertile with no obvious phenotypic abnormalities.

To study the combined effects of decreased C-P4H-I activity and lack of C-P4H-II activity, we generated P4ha1^+/−;P4ha2^−/− mice. Genotyping (Fig. 1C) of the offspring from the P4ha1^+/−;P4ha2^+/− × P4ha2^−/− matings (n = 470) indicated that the number of P4ha1^+/−;P4ha2^−/− pups was lower than expected for normal Mendelian inheritance (12% versus 25%) (Table 3). The heterozygous or homozygous inactivation of the P4ha1 and P4ha2 genes, respectively, in the P4ha1^+/−;P4ha2^−/− pups and their littermates obtained from the P4ha1^+/−;P4h2^+/− × P4ha2^−/− matings was verified at the mRNA level by RT-PCR (data not shown) and qPCR analysis of RNA isolated from cultured primary growth plate chondrocytes (Fig. 1D) or crude growth plates (data not shown). No statistically significant changes were detected in the expression level of the P4ha3 gene coding for the α(III) subunit of C-P4H-III (8), although there was a trend for a slight increase in the P4ha2^−/− and P4ha1^+/−;P4h2^−/− mice, indicating potential partial compensation (Fig. 1D).

The amount of total C-P4H activity was measured using Nonidet P-40-soluble lysates from the chondrocyte homogenates as sources of the enzyme and [¹⁴C]proline-labeled type I protocollagen as a substrate. The amount of total C-P4H activity was reduced in the P4ha1^+/−;P4ha2^−/− chondrocytes to ∼35% of that in the wild-type cells, the C-P4H activity of the P4ha2^−/−, P4ha1^+/−;P4ha2^+/− and P4ha1^+/+;P4ha2^+/− chondrocytes being 55–75% of that of the wild type (Fig. 2).

P4ha1^+/−;P4ha2^−/− Mice Display Impaired Skeletal Growth and Kyphosis Postnatally

Neither P4ha1^+/−;P4ha2^−/− (Fig. 3A) nor P4ha2^−/− mice had any overt skeletal phenotype at birth, and their weights were similar to those of the control littermates (data not shown). To measure their growth rate, the mice were weighed weekly from 5 to 20 weeks. The weights of the P4ha1^+/−;P4ha2^−/− mice were significantly lower than those of the P4ha2^−/− mice and control littermates throughout the measurement period (Fig. 3B) and a similar difference persisted throughout their life. The weights of the P4ha2^−/− mice did not differ significantly from the control littermates (Fig. 3B). The reduced postnatal body weight of the P4ha1^+/−;P4ha2^−/− mice was accompanied by moderate shortening of the long bones. The hind limbs were measured by μCT to determine potential differences in the femoral and tibial lengths in newborn (P1) and 12-week-old P4ha1^+/−;P4ha2^−/− mice relative to P4ha2^−/− and control littermates. At 12 weeks, both the femurs and tibias were significantly shorter in the P4ha1^+/−;P4ha2^−/− mice, whereas no differences were observed between the P4ha2^−/− and control mice (Fig. 3C). Also, the ratio of the femoral to tibial length was slightly, but significantly, decreased in the 12-week-old P4ha1^+/−;P4ha2^−/− mice relative to the P4ha2^−/− and control mice (83% in P4ha1^+/−;P4ha2^−/− mice versus 85–86% in P4ha2^−/−, P4ha1^+/−;P4ha2^+/− and P4ha1^+/+;P4ha2^+/−, p < 0.01, n = 5–7 per genotype) (Fig. 3C). The distorted femur/tibia length ratio was present already at newborn (P1) mice (79% versus 82–83% in the controls, p < 0.05, n = 6 per genotype) (Fig. 3C). This suggests that the growth defect is initiated already during embryonic development.

The P4ha1^+/−;P4ha2^−/− mice, but not the P4ha2^−/− or control mice, developed kyphosis at a later stage. Kyphosis, which is observed in many mouse models of chondrodysplasia upon aging, was clearly observed in the P4ha1^+/−;P4ha2^−/− mice analyzed at 48 weeks of age (Fig. 3D), whereas it was absent in 12-week-old mice, suggesting progressive development of the condition. The kyphotic index of the P4ha1^+/−;P4ha2^−/− mice differed significantly from those of the P4ha2^−/− mice and control littermates at 48 weeks of age (2.33 ± 0.32 versus 3.23 ± 0.57–3.53 ± 0.91, p < 0.05, n = 5–6).

An Inner Cell Death Phenotype Is Present in Developing P4ha1^+/−;P4ha2^−/− Growth Plates

Because of the shortened long bones of the P4ha1^+/−;P4ha2^−/− mice and because C-P4H-II has its highest expression level in chondrocytes, the limb buds and tibial epiphyseal growth plates of the P4ha1^+/−;P4ha2^−/− and P4ha2^−/− mice were histologically analyzed by comparison with the control double heterozygous mice. Hematoxylin and eosin staining and in situ hybridization for Col2a1 mRNA on histological sections of limb buds isolated from E13.5 P4ha1^+/−;P4ha2^−/−, P4ha2^−/− and the control P4ha1^+/−;P4ha2^+/− mice showed normal chondrogenic differentiation in the mutant limb buds (Fig. 4). At later developmental stages, however, a hypocellular area in the middle of both the columnar proliferative and hypertrophic zones was observed in the developing growth plates of the long bones of P4ha1^+/−;P4ha2^−/− mutant mice (Fig. 5). This phenotype was clearly detectable from E17.5 onwards, being most obvious in the newborn P4ha1^+/−;P4ha2^−/− mice, but had virtually resolved by the postnatal age of 7 days (Fig. 5). Hypocellularity in the inner region of the P4ha1^+/−;P4ha2^−/− mutant growth plates was clearly observed also in in situ hybridization analysis with riboprobes that detect the cartilaginous marker Col2a1, Col10a1, and osteopontin (Spp1) mRNAs (Fig. 6A). Hypocellularity was also present in the growth plates of E17.5 and newborn P4ha2^−/− mice, although the defect was much milder than in the P4ha1^+/−;P4ha2^−/− mice (Figs. 5 and 6A).

Next, TUNEL assay was performed on tibial sections of newborn mice to establish whether the hypocellularity in the inner region of the developing growth plate was a consequence, at least in part, of cell death. Chondrocytes normally proliferate and do not undergo cell death in the proliferative/prehypertrophic zones of the growth plate. TUNEL-positive chondrocytes were detected only in the periosteum and at the chondro-osseous junction but not in the proliferative/upper hypertrophic zones of P4ha2^−/− and control P4ha1^+/−;P4ha2^+/− mice (Fig. 6B). In contrast, dying chondrocytes were observed in the inner region of both the proliferative and hypertrophic zones of the P4ha1^+/−;P4ha2^−/− growth plates, indicating that cell death could play a role in the hypocellularity. The number of TUNEL-positive cells was low, typically 3–4 dying cells per section (Fig. 6B), which is in good accordance of the cell death rate reported earlier in α10 and β1 integrin-deficient cartilage (23, 24). Notably, dying cells were limited to the most hypoxic region of the developing growth plate.

To investigate the possibility that an impairment of chondrocyte proliferation may also contribute to the inner hypocellularity of the developing P4ha1^+/−;P4ha2^−/− growth plates, BrdU incorporation assay was performed on histological sections of the proximal epiphysis of tibiae isolated from E18.5 P4ha1^+/−;P4ha2^−/−, P4ha2^−/−, and control P4ha1^+/−;P4ha2^+/− mice. No significant difference in the proliferation rate of round proliferative chondrocytes was observed in the mutant epiphysis versus the control (Fig. 6C). As expected, the proliferation rate of the columnar proliferative chondrocytes in the growth plate was significantly higher than that of the round proliferative chondrocytes in P4ha1^+/−;P4ha2^+/− control specimens (Fig. 6C). Proliferation rate of the columnar chondrocytes appeared to be mildly reduced in the P4ha1^+/−;P4ha2^−/− and P4ha2^−/− mutants relative to the controls, but the difference was on the limit of statistical significance (Fig. 6C). We also analyzed the proliferation rate of columnar proliferative chondrocytes in 3-week-old mice. No statistically significant difference in the proliferation rates was observed between the genotypes (Fig. 6D). Therefore, the inner hypocellularity is not caused by differences in the proliferation rate.

Taken together, our findings indicate that severe deficiency in C-P4H activity in embryonic and perinatal cartilages causes increased apoptosis of the proliferating chondrocytes, resulting in an inner cell death phenotype with hypocellular areas in the developing growth plate. This phenomenon, however, is transient because hypocellularity cannot be detected at later stages of postnatal life. Interestingly, chondrocyte proliferation and hypertrophic differentiation were not affected by the impairment of C-P4H activity. Consistent with this conclusion, no differences were observed in the expression levels of the Col2a1, Col9a1, Aggrecan, and Sox9 mRNAs analyzed by qPCR (Fig. 7).

Mutations in ECM constituents have been shown to affect cell proliferation, apoptosis, migration, and differentiation via two major mechanisms, ER stress caused by impaired secretion and intracellular accumulation of the mutant molecules and impaired biomechanical properties caused by abnormal assembly of the ECM (25–27). We next analyzed the contribution of these molecular mechanisms to the observed growth plate phenotype of the P4ha1^+/−;P4ha2^−/− mice.

P4ha1^+/−;P4ha2^−/− Mutant Chondrocytes Do Not Display Any Detectable Signs of Uncompensated ER Stress

Collagen molecules that are not correctly folded are typically not secreted but are retained within the ER and degraded via an ER-associated lysosomal pathway. On the other hand, excess intracellular accumulation of unfolded/misfolded collagen may result in uncompensated ER stress that elicits an unfolded protein response. It has been proposed that unfolded protein response may play a key role in the pathogenesis of chondrodyplasias (25, 28, 29). The cell death phenotype observed in the P4ha1^+/−;P4ha2^−/− mutant growth plates was most pronounced in the inner, hypoxic region of the developing growth plate and logically reflects the fact that C-P4H requires molecular oxygen in its reaction (3, 4). Hypoxia-inducible factor (HIF)-1 is the key mediator of cellular adaptation to hypoxia and an essential survival factor for growth plate chondrocytes in vivo and inactivation of HIF-1 in cartilage leads to a severe chondrocyte death phenotype in the growth plate (30, 31). Notably, HIF-1 up-regulates C-P4Hs in chondrocytes cultured in hypoxia to ensure sufficient hydroxylation of collagens in hypoxic conditions (14, 31), and inactivation of HIF-1 in the growth plate leads to reduced hydroxylation, intracellular retention of type II collagen, and induction of ER stress (31). To assess whether ER stress was triggered in P4ha1^+/−;P4ha2^−/− or P4ha2^−/− mutant growth plate chondrocytes, we investigated the relative expression levels of BiP and Chop with qPCR in whole, crude growth plate samples isolated from newborn mice. No induction of either marker was detected in the mutant samples relative to controls (Fig. 8, A and B). Furthermore, C-P4H deficiency had no effect on Chop mRNA level in isolated growth plate chondrocytes cultured in 21% O₂, nor did it have any statistically significant additive effect on the induction of Chop when the cells were cultured in 1% O₂ for 24 h (Fig. 8C). Consistent with these findings, electron microscopic analyses of proliferative chondrocytes in the growth plate of the P4ha1^+/−;P4ha2^−/− and P4ha2^−/− mice showed normal cell morphology and well organized ER without signs of collagen accumulation within the ER lumen (Fig. 8D). Collectively, no signs of uncompensated ER stress were detected in the P4ha1^+/−;P4ha2^−/− or P4ha2^−/− mutant chondrocytes either in vivo or in vitro, and uncompensated ER stress can thus not be the underlying cause of the observed cell death phenotype.

Altered Structural and Biomechanical Properties of the ECM in Vivo Are Accompanied with Impaired Thermal Stability of Collagen Fibrils Extracted from P4ha1^+/−;P4ha2^−/− Cartilage

We next asked the question whether the reduced C-4PH activity and the cell death phenotype observed in the growth plates of the P4ha1^+/−;P4ha2^−/− mutant mice is associated with abnormalities in the ECM. Crude growth plate samples from newborn P4ha1^+/−;P4ha2^−/− and P4ha2^−/− mice and control P4ha1^+/+;P4ha2^+/− and P4ha1^+/−;P4ha2^+/− mice were analyzed by amino acid analysis to determine the 4Hyp level. The degree of proline hydroxylation, 4Hyp/(4Hyp + Pro), was significantly reduced in the growth plate in both the P4ha1^+/−;P4ha2^−/− and P4ha2^−/− mice relative to the control mice, the reduction in the P4ha1^+/−;P4ha2^−/− mice being more severe, 13–16% (Fig. 9A). Because of the reduced amount of 4Hyp, the thermal stability of collagen in these mice is likely to be reduced. The thermal stability of extracted and pepsin-digested fibrillar growth plate collagen (mainly type II collagen) was determined by digestion with a mixture of trypsin and chymotrypsin after heating to various temperatures. The T_m (midpoint of thermal transition from helix to coil) of the growth plate collagen was reduced in both the P4ha1^+/−;P4ha2^−/− and P4ha2^−/− mice relative to the controls (Fig. 9B), the reduction being ∼2 °C in the former and 1 °C in the latter. We also analyzed the growth plate collagen for possible overglycosylation of hydroxylysines. SDS-PAGE analysis showed no retardation in the mobility of collagen extracted from the mutant growth plates relative to the control (Fig. 9C), indicating that there is no major delay in the secretion of type II collagen in the mutant growth plate chondrocytes.

To analyze structural and biomechanical properties of the developing growth plates in detail, we employed histochemical stainings, transmission electron microscopy, and indentation-type AFM to analyze growth plate ECM in detail. Masson's trichome staining (Fig. 10A) and picrosirius red staining visualized by polarized light resulting in birefringence from collagen (Fig. 10B) showed abnormal accumulation of collagenous material in the central growth plate, the area where chondrocyte death occurs, in the P4ha1^+/−;P4ha2^−/− mice. In transmission electron microscopy analysis, a regular meshwork-like structure formed by collagen fibrils with a uniform thickness was observed in the control (P4ha1^+/+;P4ha2^+/−) mice (Fig. 10C). In contrast, in P4ha1^+/−;P4ha2^−/− mice, this characteristic arrangement was partially lost, and several longer and thicker fibrillar collagen molecules were present (Fig. 10C). AFM height images at the central region of the growth plate revealed flattened chondrocytes nicely organized into columns in the control newborn P4ha1^+/+;P4ha2^+/− mice and also in the P4ha2^−/− mice, whereas chondrocytes were more sparse, rounded, and less organized in the P4ha1^+/−;P4ha2^−/− mice (Fig. 10D). Furthermore, high resolution deflection images of the interterritorial matrix demonstrated a collagen network that was less dense and organized in the P4ha1^+/−;P4ha2^−/− mice compared with control and P4ha2^−/− mice (Fig. 10D). Indentation measurements using nanometer-scaled pyramidal tips within the interterritorial matrix resulted in a bimodal nanostiffness distribution (Fig. 10E), where the first low modulus peak likely depicts the proteoglycan moiety, and the second peak represents the collagen fibrils (32). The frequency distributions of the elastic moduli were comparable between the control and P4ha2^−/− mice (Fig. 10E). In contrast, the interterritorial matrix of P4ha1^+/−;P4ha2^−/− mice was significantly softer evidenced by the shift of both nanostiffness peaks. The mean values of the peaks were reduced by ∼40–50% relative to control, indicating that both the proteoglycan and collagen networks are softer in the P4ha1^+/−;P4ha2^−/− mutant growth plates (Fig. 10E). The range of the stiffness values was also reduced in the P4ha1^+/−;P4ha2^−/− mice (9.07–70.56 kPa) relative to control (14.26–107.62 kPa) and P4ha2^−/− (16.02–119.36 kPa) mice, further suggesting biomechanical impairment of the P4ha1^+/−;P4ha2^−/− growth plate cartilage. In addition, the indentation type AFM data from the peripheral growth plate, which is less hypocellular and more organized in the P4ha1^+/−;P4ha2^−/− mice when compared with the central area, showed the same tendency (data not shown), demonstrating that the biomechanical weakening is characteristic for the whole growth plate region. Taken together, these findings suggest that the severe abnormalities observed in the ECM of P4ha1^+/−;P4ha2^−/− growth plates are likely to be an important pathogenetic event in the development of chondrodysplasia in the P4ha1^+/−;P4ha2^−/− mutant mice.