| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Published online before print April 26, 2007
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
From the Laboratory of Molecular Neurosciences,* Center of Molecular Medicine Cologne, Center of Biochemistry, Cologne; the Center of Anatomy, Faculty of Medicine, University of Cologne, Cologne; and the Institute of Biomechanics and Orthopaedics, Deutsche Sporthoch-schule Köln, Cologne, Germany
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
We have generated the smpd3-null mouse, the phenotype of which is characterized by the impaired secretion of peptide releasing hormones from hypothalamic neurosecretory neurons. Strong perturbation of the hypothalamus-pituitary growth axis and reduction of the number of pituicytes in the anterior lobe of the pituitary in smpd3–/– mice led to a novel form of late embryonic-juvenile combined pituitary hormone deficiency. Reduced growth hormone (GH) production and low-serum insulin-like growth factor (IGF) 1 concentrations caused a prolongation of the cell cycle in all tissues and led to a generalized hypoplasia. The most striking marker of the smpd3–/– mouse is the embryonic and juvenile dwarf phenotype associated with chondrodysplastic bone and joint deformations. We observed a low expression of smpd3 also in differentiating chondrocytes in the epiphyseal growth zone.
To determine the role of SMPD3 in ossification and longitudinal growth of long bones, we have rescued the smpd3–/– mouse from dwarfism and skeletal deformations by introducing the smpd3 cDNA as a transgene into smpd3–/– mice. The Col2a1 promoter was used for regulated cell-specific expression of the transgene in the smpd3–/– mouse. The smpd3 transgene rescued not only the hypothalamus-pituitary growth axis, normal body weight, and growth but also long bone and joint structure. Our experiments suggest the essential systemic and cell-specific role of SMPD3 in the regulation of normal growth and skeletal development.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
The smpd3–/– and smpd2/3 double knockout mutant mouse lines used in this study have been previously described.6 Genotypes were assessed by polymerase chain reaction (PCR) and Southern blot hybridization analysis of tail DNA. Homozygous newborn were recognized unambiguously by rhizomelic short, severely deformed fore and hind limbs. The wild-type, smdp3+/–, and smpd3–/– mice used in this study were offspring derived from intercrosses between heterozygous smpd3+/– mice on a C57BL/6129Sv.
Smpd3 Transgenic Rescue of the smpd3–/– Phenotype
Full-length smpd3 cDNA fused in frame with green fluorescent protein at the 3' end was ligated blunt-ended into the dephosphorylated EcoRV site of the 6.2-kb col2A1 promoter. The 9-kb transgene sequence was released from the plasmid by BssHII restriction enzyme digestion, subjected to gel electrophoresis, and purified on a Nucleospin column (Macherey & Nagel, Düren, Germany) as a prelude to pronuclear injection into fertilized oocytes from smpd3–/– females.
Genotyping of Transgenic smpd3 Mice of Founders and Offspring
The mutated smpd3locus (smpd3–/–) and the transgene of founders and offspring were characterized by PCR using genomic DNA (0.25 to 1 µg). Smpd3homozygosity of the founder mice was checked by PCR using primers smpd3 untranslated region 5' (5'-TGCATGATGAGAGTCTGGGTCCAGACCTGC-3') annealing to 5' noncoding sequences external of the targeting construct and smpd3ex1as (5'-CTTGAGAAACAGACCTCCCTTAGAGGCCAG-3') (expected band size: 3.3 kb). The correct integration of the smpd3-egfp rescue cDNA was confirmed with 5' forward primer Col2a1proms (5'-TCCTCACCTCCAGCGATATTAGCGCCGCTG-3') and the smpd3exIas (5'-CTTGAGAAACAGACCTCCCTTAGAGGCCAG-3') (expected band size: 1.6 kb). The integration of the complete transgene was confirmed with 5' forward primer Col2a1proms (5'-TCCTCACCTCCAGCGATATTAGCGCCGCTGG-3') and Col2a1promas (5'-AGCAGGAGGTGTTTGACACAGAATAGCACC-3') (expected band size: 3.3 kb).
The endogenous col2A1 promoter was amplified by using primer col2A1 prom and Col2A1 rev primer, both annealing to the promoter col2A1. PCR fragments were analyzed in 1.0% agarose gels containing ethidium bromide.
RNA Isolation from Bone and Semiquantitative Reverse Transcription (RT)-PCR
Long bones (femur and tibia) were freed from soft tissues and immediately powderized under liquid nitrogen. Total RNA was isolated for Northern blot analysis by the TRIzol method (Invitrogen, Carlsbad, CA) and poly(A)-mRNA using the Oligotex mRNA Midi Kit (Qiagen, Hilden, Germany) following the manufacturers’ recommendations. Quality-controlled RNA was transcribed using mouse leukemia virus reverse transcriptase (Invitrogen). From the total RNA samples isolated by the TRIzol method, between 5 and 10 µg isolated from different tissues were reverse transcribed into cDNA. cDNA aliquots and specific smpd3 and hypoxanthine-guanine-phosphoribosyl transferase (hgprt) sense and antisense primers were used in quantitative PCR amplification. PCR reactions were optimized for each primer pair at 15, 20, 25, and 30 cycles to ensure the linear range and were performed in the presence of 1 µCi of [
-32P]dCTP (1 Ci, 37 GBq). PCR fragments were analyzed on 6% polyacrylamide gels run in sodium borate buffer and then transferred to Whatman filters (Clifton, NJ). Radioactive signals were detected by phosphorimaging, and bands were quantified by densitometric scanning using the IMAGE Quant software (GE Healthcare, Little Chalfont, Buckinghamshire, UK). The signals of smpd3 were normalized to hgprt cDNA levels.
Protein Analysis of Bone Tissue
Long bones of wild-type and smpd3–/– mice were powderized under liquid nitrogen, demineralized by dialysis against 10% acetic acid for 24 hours, and centrifuged at 13,000 rpm, and the sediment was incubated overnight in 0.5 mol/L acetic acid containing 1 mg of pepsin per 20 mg of bone. The turbid solution was cleared by centrifugation at 13,000 rpm for 10 minutes, and the supernatant was used for gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (4 to 12%). All steps were performed at 4°C.
Neutral Sphingomyelinase Assay
Neutral SMase (SMPD2 and SMPD3) activity was determined in protein fractions of the Triton X-100 solubilized 100,000 x g sediment of the postmitochondrial fraction and acid sphingomyelinase (SMPD1) activity in the sediment of the 12,000 x g fraction using N-[14CH3]-sphingomyelin as a substrate.2,4
Immunohistochemistry and Histology
Freshly prepared long bones (femur, tibia, and humerus) were fixed in 4% buffered paraformaldehyde overnight and embedded in the methylacrylate polymerization system Technovit 9100 NEU (Heraeus Kulzer, GmbH & Co, Wehrheim, Germany) following the manufacturer’s instructions. For immunohistochemistry, 5-µm sections were processed followed by examination under a light microscope. Mouse anti-cartilage oligomeric matrix protein (COMP) and anti-matrilin 3 antibodies were kindly provided by Dr. R. Wagener, Center of Biochemistry, Cologne, Germany. Anti-osteopontin was purchased from Assay Designs, Inc. (Ann Arbor, MI). Cy 3-labeled secondary anti-rabbit antibody was used for immunofluorescence microscopy.
Pituitaries of 18-day-old wt and smpd3–/– mice were perfused with 4% buffered paraformaldehyde from the left ventricle and embedded, and 5-µm sections were obtained for immunohistochemistry of GH-, thyroid stimulating hormone-, follicle stimulating hormone-, and luteinizing hormone-producing pituicytes. Pituitary hormone antibodies were kindly supplied by Dr. A.F. Parlow, National Hormone and Pituitary Program, National Institute of Diabetes and Digestive and Kidney Diseases, Torrance, CA; anti-GH-releasing hormone was provided by Dr. G. Thordarson, University of California Santa Cruz, Santa Cruz, CA.
Peripheral Quantitative Computed Tomography (pQCT)
Right and left femora of 2-, 7-, and 20-month-old wild-type and smpd3–/– mice were scanned by pQCT using the XCT Research M scanner and version 5.50 of the software (Stratec Medizintechnik GmbH, Pforzheim, Germany). For the measurements, isolated bones were placed, with the anterior surface upwards, in a syringe filled with saline solution. After scout view, sections were made at the distal femoral metaphysis (at 15, 17.5, and 20% of total bone length measured from the distal joint line) and at the midshaft (at 50% of total bone length). The voxel size was 500 x 70 x 70 µm. Each slice was analyzed by contour mode 1, peel mode 20 (30%), and cortical mode 1 (710 mg/cm3). At the femoral metaphysis, total cross-sectional bone area (CSA, mm2), total bone mineral density (BMD, mg/cm3), total bone mineral content (BMC, mg), trabecular CSA (Tb.CSA, mm2), trabecular BMD (Tb.BMD, mg/cm3), and trabecular BMC (Tb.BMC, mg) were determined as the mean of three slices. At the mid-diaphysis, the cortical area (Ct.CSA, mm2), the cortical BMD (Ct.BMD, mg/cm3), the cortical BMC (Ct.BMC, mg), the cortical thickness (Ct.thickness, mm), the periosteal circumference (mm), and the endosteal circumference (mm) were evaluated. Reproducibility of pQCT measurements with the above settings was determined by repeated scans of mouse femora with repositioning. The root-mean square average CV% values were 2.2% for Tb.BMD and 0.7% for Ct.BMD.
Radiography
Anesthesized wild-type and smpd3–/– littermates were examined using a bench X-ray unit (HP Cabinet X-ray System-Faxitron series, model 43855A; Hewlett-Packard, McMinnville, OR), with single-side emulsion film (Agfa-Ts Structurix D4DW, NDT System; Grosche, Bottrop, Germany) at 40 kV with exposure times of 25 seconds for young and 50 kV and 48 seconds for adult mice.
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
The most striking markers of the embryonic-juvenile hypoplasia phenotype of the smpd3–/– mouse are the retarded longitudinal growth of bones, increased mineralization, and massive malformation of long bones and joints. Here, we investigated the role of smpd3 expression during the development and growth of the skeletal system and the impact of the loss of smpd3 expression in the SMPD3-deficient mouse mutant, which was generated by homologous recombination using a targeting construct, in which exon I of the smpd3 gene was disrupted (Figure 1A)
. Semiquantitative RT-PCR of multiple tissue RNA demonstrated that smpd3 is ubiquitously expressed with highest levels in the central nervous system and the immune system but low levels in bone (Figure 1B)
. In the mutant mouse, no smpd3 transcripts were detectable by RT-PCR in total mRNA of long bones (Figure 1C)
.
|
. In the smpd3–/– mutant mouse, the residual activity was attributed to SMPD2. Note that SMPD2 activity was completely abolished in bone protein extracts of the smpd2/smpd3 double knockout mutant (Figure 1D)
.
The early onset of the retarded growth leading to the dwarf phenotype was revealed by comparative X-ray imaging of the skeleton of smpd3–/– embryos of age e16, p2, and p18, as well as juvenile and 18-month-old adult mutant mice. Severe short-limb dwarfism with pronounced joint deformation was observed during the early postnatal growth phase when pressure and traction are exerted on femur, tibia, humerus, and the thoracic and lumbar vertebra (Figure 2, A–F)
. The articular ends of femur, tibia, and humerus are bossed, and elbow and knee joints are restricted in rotation and extension. The lateral view clearly shows kyphosis in the thoracolumbar area. Smpd3–/– mice also have a narrow long trunk and a disproportionately large head (Figure 2A)
. Moreover, X-ray imaging indicated enhanced calcification of ossified bones of smpd3–/– mice beyond 2 months of age, which was quantified here by pQCT (Figure 2, E and H)
. Note that in the smpd3–/– mouse mutant, incisors were normally formed, and no fractures or callus formation were observed.
|
. BMD was higher at the metaphysis of smpd3–/– mice than in control: 543 ± 15 versus 426 ± 6 (2 months), 535 ± 14 versus 428 ± 13 (7 months), and 546 ± 2 versus 354 ± 1 (20 months), and likewise ctBMD at the mid-diaphysis in agreement with the X-ray data. Therefore a lack of SMPD3 seems not to affect mineralization of bone. Further, the periosteal and endosteal circumferences appear to be similar (Figure 2G)
. Because sphingomyelin metabolism and storage in the SMPD1-deficient Niemann-Pick mouse are known to be severely affected,4 we performed lipid (sphingomyelin, sphingosine, and ceramide) analyses of long bones of control and smpd3–/– mice. Examination of the total lipid profiles revealed no storage of sphingomyelin in smpd3–/– mice. Unlike the SMPD1-deficient mouse,4 macrophages of the smpd3–/– bone marrow showed no sphingomyelin accumulation, and the ceramide concentration also remained unchanged. The absence of sphingomyelin storage in other reticuloendothelial tissues has been shown previously.6 Therefore, we conclude that nSMase deficiency has no impact on the lipid composition in bones in general and on sphingomyelin and ceramide metabolism and storage in particular.
We next studied the organization of the epiphyseal growth plate of the proximal tibia of wild-type and smpd3–/– mice by immunohistochemistry. The proximal tibia of smpd3–/– mice showed a narrow and disorganized growth zone, irregular columnization of cartilage with short rows of small cells, and reduced hypertrophic cells columns. Primary trabeculae were thick, irregularly arranged, and scarcely calcified. SPMD3, collagen, and three noncollagenous proteins abundant in the cartilage extracellular matrix of the epiphyseal growth plate, COMP,7-9
matrilin 3,10,11
and osteopontin12
were also studied. In wild-type p20 mice, COMP is uniformly distributed throughout the interterritorial extracellular matrix of cartilage. However, even at p20 smpd3–/– mice, COMP immunostaining of cartilage was still restricted to the immediate pericellular matrix of the chondrocytes similar to the COMP distribution in human fetal cartilage.9
Large longitudinally oriented and tightly aligned islets of cartilage extend into the ossification zone of long bones in age- and gender-matched control mice, whereas in the smpd3–/– mutant, layers of COMP and matrilin-positive small and irregularly shaped chondrocytes form a sharp boundary toward the ossification zone (Figure 3, A and B)
. Osteopontin-reactive material surrounded large chondrocytes ordered in columns in the growth plate of control mice, whereas in the smpd3–/– mouse, irregularly packed small chondrocytes were covered with osteopontin (Figure 3C)
. Severe disorganization of collagen bundles between chondrocytes was also observed in the smpd3–/– mutant mouse (Figure 3E)
. These data indicate that the delayed maturation of chondroblasts together with an abnormal extracellular arrangement of noncollagenous proteins and of collagen in the extracellular matrix of cartilage during late embryonic and juvenile development delays the longitudinal growth of long bones and leads to diaphyseal and joint deformations. Smpd3 is also expressed locally in chondrocytes in the epiphyseal growth zone (Figure 3D)
. Therefore, SMPD3 deficiency in differentiating chondrocytes might contribute to the skeletal phenotype. In smpd3–/– mice, bones are deformed, but no fractures occur, and mineralization is normal. We did not observe any SMPD3-immunoreactive protein in the growth plate and the primary ossification center or the calcified shaft of long bones of smpd3–/– mice (Figure 3D)
.
|
The promoter of the Col2A1 gene is known to induce chondrocyte-specific expression. Therefore, high-level expression would be expected in cartilaginous tissues although low-level expression has also been observed in extraskeletal locations, such as the developing brain13 .14
To this end, full-length smpd3 cDNA was fused with enhanced green fluorescent protein and inserted 3' to the Col2a1 promoter.14
The Col2a1-smpd3-egfp gene construct carried the 5' sequences of the mouse type II collagen promoter as described before14
(Figure 4A)
. A 9-kb BsstII fragment free of vector sequences was used for pronuclear microinjection into fertilized smpd3–/– oocytes. The mutated smpd3 locus and the transgene of founders and offspring were characterized by PCR (Figure 4, B–E)
. The primers and their sequences are listed under Materials and Methods. A total of four independent founders were obtained, and the copy number of the transgene determined by semiquantitative PCR as described under Materials and Methods. Founders carried copy numbers between three and six. Founder 5, with six copies of the smpd3 transgene, was used to establish a stable transgenic line. In the smpd3–/– rescued mouse, the smpd3 transgene was expressed in most organs, once again strongest in brain, but also in bone. Semiquantitative RT-PCR indicated ubiquitous smpd3 expression, strongest in brain, jejunum, kidney, thymus, and bones. (Figure 4F)
. Comparative immunohistochemistry of the hormone-secreting pituicytes in the anterior pituitary lobe of control wt, smpd3–/–, and rescued smpd3–/– mice (age p15), using GH, thyroid-stimulating hormone, follicle-stimulating hormone, and luteinizing hormone antibodies, documented the return of the anterior pituitary secretory function and demonstrated that, secondary to the rescue of smpd3 expression, a high level of correction of the combined pituitary hormone deficiency had occurred (Figure 5)
.
|
|
X-ray imaging revealed a regular morphology of long bones of fore and hind legs in the smpd3–/– rescued mouse (Figure 4, G–J)
. The development and organization of epiphyseal growth plates were normalized. Moreover, the smpd3 transgene restored the regular morphology of the epiphyseal growth plate (Figure 4, K–N)
. Regulated expression of the transgene under the control of the Col2a1 promoter not only rescued normal longitudinal growth and ossification of long bones, as well as abolishing chondrodysplastic deformations, but also rescued normal systemic growth. Thus, our rescue experiments establish the essential systemic and cell-specific role of SMPD3 in the regulation of skeletal development as well as normal growth. Taken together, the present study provides substantial evidence for an important role of SMPD3 in normal chondrocyte differentiation and enchondral ossification in the growth plate of long bones during late embryonic and postnatal and juvenile development, essential for regular longitudinal growth.
Malfunctioning of the hypothalamus-pituitary axis in smpd3-null mice perturbs the pituitary-epiphyseal growth plate axis because of the low GH production by the reduced number of somatotrophic pituicytes.8 GH controls chondrocyte proliferation and IGF1 chondrocyte differentiation.15 GH exerts indirect effects via IGF, but the two hormones, GH and IGF1, also exert direct local effects on epiphyseal chondroblasts in the germinal zone, and, in addition, IGF1 enhances the development to hypertrophic chondrocytes.16,17
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
The smdp3–/– mouse also differs significantly from the "fro" (fragilitas ossium) mouse, which was isolated from a randomly bred stock of mice generated by chemical mutagenesis with the mutagen Tris(1-aziridinyl) phosphine-sulfine. fro is an autosomal recessive mutation with high lethality assigned to the deletion of a 1.5-Mb chromosomal segment in the midpart of mouse chromosome 8.19 Recently, intron 8 and most of exon 9 of the smpd3 gene were reported to be part of this deletion.20
The fro phenotype is characterized by osteoporosis due to enhanced osteoclast activity, multiple fractures of the long bones and ribs, diaphyseal deformations, thin cortices, calluses, short stature, brittle teeth, and reduced osteoblasts. fro–/– mutant mice appear to have normal cartilage growth despite exhibiting hypomineralization. Loss of local SMPD3 activity and defective sphingomyelin hydrolysis, as well as disruption of the ceramide pathway including sphingosine-1 phosphate, have been proposed to affect bone mineralization and lead to bone fragility.20 Because the fro/fro mouse shares symptoms with a severe, recessive form of human osteogenesis imperfecta, it has been proposed as a mouse mutant model for this disease.19-23 The fro and smpd3–/– mutant mice described here have contrasting phenotypes. Maturation of chondrocytes in the epiphyseal growth plate of our smpd3–/– mice is severely perturbed, mineralization of long bones of juvenile smpd3–/– mice is normal and, in adult null mice, significantly stronger than in control animals. Therefore, these hallmarks of the smpd3–/– mutant mouse offer phenotypic markers that differ from those for the fro–/– mouse. Ceramide and sphingosine-1 phosphate, two metabolites of sphingomyelin catabolism missing in the fro–/– mouse, have been proposed as signaling molecules for the hypermineralization of the osteogenesis imperfecta phenotype. However, analytical data of bone lipid extracts of the fro–/– mutant are currently not available. Interestingly, the lipid composition (including sphingomyelin, ceramide, and sphingosine) in bone extracts of control, smpd3–/–, and the double mutant smpd2/smpd3–/– mice, completely devoid of neutral sphingomyelinase activity, was identical. The contrasting phenotypes of the fro–/– and smpd3–/– mutant mice can hardly be explained by differences in the genetic background of the two mouse lines. The background of the smpd3–/– mouse is C57BL/6x129Sv, which has been backcrossed for more than 10 generations into C57BL/6.
The question therefore arises as to whether the smpd3 deletion in the fro–/– mouse induced by chemical mutagenesis is of a random nature and only one symptom, and that the manifesting phenotype is not due specifically to smpd3 deletion, but rather to the whole gamut of point mutations, insertions, deletions, and rearrangements in the genome, superimposing the molecular pathology of the smpd3deletion by a variety of additional unknown molecular events. In other words, this observation may be due to complex mutagenesis masking the specific effect of deletion of smpd3 alone. This hypothesis can only be clarified by a broad and comprehensive genetic analysis of the fro–/– mouse mutant.
|
Footnotes |
|---|
Supported by the Center for Molecular Medicine Cologne (CMMC), the Deutsche Forschungsgemeinschaft (Sto32/38-2), and the Gunther and Arina Lauffs Foundation.
The authors declare that they have no competing financial interests.
Accepted for publication March 15, 2007.
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
