Upregulation of cGMP-dependent Protein Kinase (PRKG1) in the Development of Adolescent Idiopathic Scoliosis.

OBJECTIVE: To explore the molecular regulatory mechanisms underlying fibroblast differentiation and dysfunction in the development of adolescent idiopathic scoliosis (AIS) in an effort to identify candidate therapeutic targets for AIS. METHODS: The GSE110359 dataset, obtained from the bone marrow stromal cells of 12 AIS patients and five healthy controls, was retrieved from the GEO database. The data were preprocessed and differentially expressed genes (DEGs) were identified. KEGG pathway and Gene Ontology (GO)-Biological Process (BP) enrichment analyses were performed to identify the function of the DEGs. A protein-protein interaction (PPI) and a microRNA-transcription factor (TF)-target co-regulatory network were constructed to identify hub genes in the development of AIS. In addition, hub DEGs were evaluated by quantitative PCR (qPCR) and immunohistochemical staining. RESULTS: A total of 188 DEGs including 100 up-regulated and 88 down-regulated genes were obtained. The up-regulated DEGs were related to "p53 signaling pathway", "FoxO signaling pathway", and "cGMP-PKG signaling pathway" terms, while the down-regulated DEGs were significantly enriched in seven terms including "protein processing in endoplasmic reticulum". The key up-regulated genes, PRKG1, CCNG2, and KAT2B, and the key down-regulated genes, MAP2K1 and DUSP6, were identified by the PPI and miRNA-TF-Target regulatory network analyses. mRNA expression patterns for PRKG1, DUSP6, and KAT2B were successfully verified by qPCR. In addition, PRKG1 protein levels were found to be elevated during the immunohistochemical analysis. CONCLUSION: Increased expression of PRKG1 in AIS patients might be an attractive therapeutic target for AIS. However, further gain or loss-of-function studies should be conducted.

Impaired Annulus Fibrosus Development and Vertebral Fusion Cause Severe Scoliosis in Mice with Deficiency of c-Jun NH2-Terminal Kinases 1 and 2.

Mitogen-activated protein kinases, including c-Jun NH2-terminal kinase (JNK), play an important role in the development and function of a large variety of tissues. The skeletal phenotype of JNK1 and JNK2 double-knockout (dKO) mice (JNK1fl/flCol2-Cre/JNK2-/-) and control genotypes were analyzed at different embryonic and postnatal stages. JNK1/2 dKO mice displayed a severe scoliotic phenotype beginning during development that was grossly apparent around weaning age. Alcian blue staining at embryonic day 17.5 showed abnormal fusion of the posterior spinal elements. In adult mice, fusion of vertebral bodies and of spinous and transverse processes was noted by micro-computed tomography, Alcian blue/Alizarin red staining, and histology. The long bones developed normally, and histologic sections of growth plate and articular cartilage revealed no significant abnormalities. Histologic sections of the vertebral column at embryonic days 15.5 and 17.5 revealed an abnormal organization of the annulus fibrosus in the dKOs, with chondrocyte-like cells and fusion of dorsal processes. Spinal sections in 10-week-old dKO mice showed replacement of intervertebral disk structures (annulus fibrosus and nucleus pulposus) by cartilage and bone tissues, with cells staining for markers of hypertrophic chondrocytes, including collagen X and runt-related transcription factor 2. These findings demonstrate a requirement for both JNK1 and JNK2 in the normal development of the axial skeleton. Loss of JNK signaling results in abnormal endochondral bone formation and subsequent severe scoliosis.

Lack of association between the promoter polymorphisms of MMP-3 and IL-6 genes and adolescent idiopathic scoliosis: a case-control study in a Chinese Han population.

STUDY DESIGN: Case-control study. OBJECTIVE: This study is to replicate the association between the promoter polymorphisms of matrix metalloproteinase (MMP)-3 (-1171 5A/6A rs3025058) and interleukin (IL)-6 genes (-174G/C rs1800795) and adolescent idiopathic scoliosis (AIS) in a Chinese Han population. SUMMARY OF BACKGROUND DATA: Recently, promoter polymorphisms in MMP-3 and IL-6 have been reported to be associated with AIS. Such genetic association, if confirmed by replication in other samples, would point to a primary degenerative defect in the disc or nucleus pulposus and inflammation as the key pathogenic mechanisms of AIS. METHODS: A total of 487 Chinese girls with AIS and 494 healthy age-matched adolescent girls were recruited consecutively during a 3-year period. The same genotyping technique as the original report was used to detect promoter polymorphisms of the MMP-3 and IL-6 genes. Statistical analysis of genotype frequencies between AIS patients and normal controls were performed by chi test. RESULTS: In this association study of the MMP-3 polymorphism and the risk of scoliosis, no significant difference was found between cases and controls, both in term of allelic association (6A: 81.2% in cases vs. 81.8% in controls, 5A: 18.8% in cases vs. 18.2% in controls, P = 0.745) or genotype association (6A/6A: 65.9% in cases vs. 66.2% in controls, 5A/6A: 30.6% in cases vs. 31.2% in controls, and 5A/5A: 3.5% in cases vs. 2.6% in controls; P = 0.733). Among AIS patients, the maximal Cobb angles were also not different among MMP-3 genotypes (6A/6A: 31.1 degrees +/- 9.7 degrees, 5A/6A: 29.1 degrees +/- 10.5 degrees, and 5A/5A: 29.4 degrees +/- 11.2 degrees; P = 0.392). As for IL-6 polymorphism, -174G/C polymorphism was not found in the Chinese AIS patients, and all 100 AIS patients and 100 normal controls were found to carry the G/G wild type. CONCLUSION: This study did not find any significant association of promoter polymorphisms of the MMP-3 (-1171 5A/6A rs3025058) and IL-6 gene (-174G/C rs1800795) with AIS. The results indicate that the MMP-3 promoter polymorphism is not associated with AIS in the Chinese population. Further studies, however, are needed to rule out the potential association with other promoter polymorphisms in IL-6.

Association study of tryptophan hydroxylase 1 and arylalkylamine N-acetyltransferase polymorphisms with adolescent idiopathic scoliosis in Han Chinese.

STUDY DESIGN: A genetic association study of tryptophan hydroxylase 1 gene (TPH1) and arylalkylamine N-acetyltransferase gene(AANAT) with adolescent idiopathic scoliosis (AIS) in Han Chinese. OBJECTIVE: To access whether TPH1 and AANAT polymorphisms are associated with the predisposition, gender, and/or severity of AIS. SUMMARY OF BACKGROUND DATA: Studies have shown that AIS is a multifactorial inheritance disease, but the etiology is still unknown. In addition, several lines of evidence show that melatonin deficiency is closely associated with AIS, although there are still doubts and debates. Some polymorphisms in TPH1 and AANAT, the genes of 2 critical enzymes involved in melatonin biosynthesis, may contribute to variability of melatonin production in pineal glands. METHODS: We genotyped 16 reported single nuclear polymorphisms (SNPs) present in TPH1 and AANAT in 103 AIS patients and 108 controls with matched sex and age. The data of 6 SNPs with minor allele frequence (MAF) above 5% were analyzed by the allelic and genotypic association analysis, the genotype-phenotype (gender and Cobb angle) association analysis, and the haplotype analysis. RESULTS: The single SNP analysis showed that rs10488682, located in the promoter region of TPH1, was related with the occurrence of AIS (P < 0.05). No SNP was found to be correlated with gender or Cobb angle. Two makers (rs8176799 and rs2108977) in TPH1 were found to be in strong LD [ D' = 1.0 (95% CI, 0.9-1.0), gamma = 0.501, LOD = 18.93] in the controls. Both global haplotype analysis and individual haplotype analysis showed that there was no haplotype significantly associated with AIS in this LD block. CONCLUSION: TPH1 polymorphisms were associated with AIS but not with gender and Cobb angle in AIS patients. AANAT polymorphisms were not associated with AIS. These results suggested that TPH1 was an AIS predisposition gene, and there was a close relationship between the dyssynthesis of melatonin and AIS.

Human platelet Ca2+-ATPases: new markers of cell differentiation as illustrated in idiopathic scoliosis.

The aetiology of adolescent idiopathic scoliosis (AIS), the most common form of scoliosis, is unclear. Previous studies showed controversial platelet abnormalities including intracellular calcium. Platelet Ca2+ homeostasis is controlled by a multi-Ca2+-ATPase system including SERCA (sarco/endoplasmic reticulum Ca2+-ATPase) and PMCA (plasma membrane Ca2+-ATPase) isoforms. Here, we first investigated the expression of PMCA4b, SERCA3a and SERCA2b isoforms in platelets of 17 patients with AIS. Patients presenting thoracic curves were found to present a higher PMCA4b expression coupled to a lower SERCA3a one in agreement with an abnormality in platelet maturation. Indeed, using PMA-treated MEG 01 cells, an in vitro model of megakaryocytopoiesis, we found an increase in SERCA3a expression, associated to a caspase-3 mediated C terminal proteolysis of PMCA4b. To look whether platelets reflect a basic defect in cell differentiation, we next identified osteoblast Ca2+-ATPases and studied their expressions in AIS. Major expressions of PMCA4b and SERCA2b were found in normal osteoblasts. Comparing platelets and osteoblasts in two additional patients with AIS, we found opposite and concerted regulations of the expressions of PMCA4b and caspase-3 substrate, PARP in both cell types. A systemic defect in cell differentiation involving caspase-3 can be proposed as a novel mechanism in the etiopathogenesis of the most frequent type of AIS. *R. Bredoux and E. Corvazier contributed equally to this work.

Cystathionine beta synthase deficiency affects mouse endochondral ossification.

Cystathionine beta synthase (CBS) is a crucial regulator of plasma concentrations of homocysteine. Severe hyperhomocysteinemia due to CBS deficiency confers diverse clinical manifestations, notably characteristic skeletal abnormalities. To investigate this aspect of hyperhomocysteinemia, we analyzed the skeleton of CBS-deficient mice, a murine model of severe hyperhomocysteinemia. Radiography, Alcian Blue/Alizarin Red S-stained whole skeletal preparations, and histological comparisons were used to determine the extent, pattern, and distribution of skeletal abnormalities in CBS-deficient mice. Disruption of the murine CBS gene leads to skeletal abnormalities, notably kyphoscoliosis, with temporal shortening of long bones due to impaired cartilage differentiation, albeit to differing degrees.

[Expression of neuronal nitric oxide synthase and inducible nitric oxide synthase in the erector spinal muscles in idiopathic scoliosis].

OBJECTIVE: To explore the possible mechanism of the erector spinal muscles in idiopathic scoliosis by comparing the expression and localization of neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) of the thoracic erector spinal muscles on convex side and concave side. METHODS: The patient group comprised 8 females and 2 males who were scheduled for spinal surgery. The apex of scoliotic curve in these patients arose between T6 and T11. The mean age was 14.3 (range 12-17) years, and the mean Cobb angle was 57.7 degrees (range 45 degrees-85 degrees). Muscle biopsies were taken bilaterally during surgery from the superficial multifidus muscle at the apex of the curve between the 6th and 11th thoracic vertebral levels. Part of the tissue was fixed in formalin and stained with hematoxylin and eosin; the remaining tissue was snap frozen and processed for immunohistochemistry and Western blot. Immunocytochemistry for nNOS and iNOS were performed using the EnVision two-step method. Western blot was done with antibodys to nNOS and iNOS. Immunoreactive bands were visualized by enhanced chemiluminescence according to the manufacturer's specifications (Amersham Corp). RESULTS: nNOS protein in the erector spinal muscles was localized at the sarcolemma. Western blot demonstrated that nNOS protein expression in the concave side of erector spinal muscles is more than that in the convex side. A significant decrease in nNOS protein and activity was found on the convex side of erector spinal muscles from idiopathic scoliosis patients; There was a little immunoreactivity to iNOS in erector spinal muscles. There was little difference in iNOS protein expression between both sides of the curve. Western blot detected the same results. CONCLUSION: There is a greater expression of nNOS and iNOS on the concave side than on the convex side, suggesting nNOS and iNOS may play a role in the pathogenesis of idiopathic scoliosis.

[Alkaline phosphatase level and bone density in Scheuermann's disease and in adolescent idiopathic scoliosis]. / Az alkalikus foszfatáz-szint és csontsúrúség Scheuermann-kórban és adolescens idiopathiás scoliosisban.

Authors made a comparison between 167 suffers from Scheuermann's disease (SD), 70 adolescence idiopathic scoliosis (AIS) and 132 age, sex, height, weight, pubertal developmental stages (Tanner stages) matched controls. The height percentile in 130 cases was also determined. The bone mineral density (BMD) was measured by pQCT on the non dominate sided radial bone. The alkaline phosphatase (AP) level increased at the beginning of puberty and in the puberty in SD. In SD the trabecular Z-score of BMD was significantly decreased in Tanner stage 1 to 4 in both boys and girls. It was not found any significance difference, however, in Tanner stage 5, while in AIS girl no significance decrease of BMD was found. In SD good correlation could be demonstrated between increase in AP and decrease in trabecular Z score r = 0.2, while did not correlate with height percentile. The AP level's increase, and radiomorphometric data of SD severity (intervertebral space narrowing and antero-posterior diameter increase of vertebral body) shows a significant correlation with decreased trabecular Z-score in the period of prepuberty.

[Histological and histochemical changes in the neck muscles of spontaneously occurring scoliosis in a special strain of Japanese quail, SQOHM].

A strain of Japanese quail, SQOHM, is known as a model of idiopathic scoliosis in humans. It shows hereditary scoliotic deformities occurring in the cervical region. Histological, histochemical and serological studies were performed in 51 scoliotic and 109 non-scoliotic quail. Histological study revealed internal nuclei and tiny groups of small fibers in the neck muscles. The incidence of these, however, was only 19% in the scoliotic and 7% in the non-scoliotic quail. Histochemical observation with adenosine triphosphatase, phosphorylase and succinic dehydrogenase stains was made on the dorsal neck muscles. An increase in the number of beta-fibers, and a decrease in the number and hyperplasia of alpha-fibers were observed on the concave side of the scoliosis. The increase in number of beta-fibers on the concave side can play a role in promoting the deformity was suggested.

Changes in histochemical profile of muscle after long-term electrical stimulation in patients with idiopathic scoliosis.

Adolescent patients with idiopathic scoliosis were treated with long-term electrical stimulation (30 Hz) at the posterior axillary line on the convex side of the curvature in order to correct the spinal deformity. The patients were also followed with muscle biopsies from the latissimus dorsi of the stimulated side taken before, after 3 and 6 months of electrical stimulation. There was a tendency for an increase in the percentage of type I and especially the type II C (undifferentiated) fibers after stimulation. The mean muscle fiber area and the fiber areas of the various fiber types did not change significantly. Histopathological findings were generally rare before as well as after 3 months of electrical stimulation, the only noticeable finding being a somewhat increased frequency of atrophic fibers in groups after 6 months of stimulation. In all studied patients the enzymatic activity of citrate synthase increased after 3 months and further in three studied patients after 6 months of stimulation. The present study gives some evidence of an adaptive process caused by electrical stimulation towards a more fatigue-resistant muscle.

Morphology of the paravertebral muscles in adolescent idiopathic scoliosis.

Histochemical studies of paravertebral muscles in idiopathic scoliosis have shown a consistently higher proportion of type I fibers on the convex side. In this study of the transversospinal muscles in moderate idiopathic scoliosis, we could demonstrate a lower type II B/II A fiber ratio on the convex side, along with an increased proportion of type I fibers. The capillary count was also higher on the convex side, especially around the type I fibers. The few pathologic changes found were predominately seen in the gravest cases of scoliosis. It is concluded that the fiber type distribution, capillary count, and metabolic enzyme activity on the convex side resembles that seen after endurance training. This suggests a secondary adaptive origin of these changes.

Association of glycosaminoglycan depletion and degradative enzyme activity in scoliosis.

Although several theories have been advanced about etiology of idiopathic scoliosis, the pathogenesis still remains unknown. One study detected a decrease in the glycosaminoglycan content of the nucleus pulposus in idiopathic scoliosis, and it was theorized that this represented increased degradation. The present study was designed to investigate degradative enzyme activity in scoliotic intervertebral disks. Twenty-three disks from 5 patients with idiopathic scoliosis and 18 disks from 3 patients with scoliosis resulting from myelomeningocele were obtained at surgery (Dwyer procedure). Five disks were obtained during 2 postmortem examinations. Analyses of hydroxyproline, hexosamine and acid phosphatase were performed separately on the annulus and nucleus of each disk. Hexosamine was decreased in idiopathic scoliotic nuclei versus controls (p less than 0.001) by approximately 25%. Hydroxyproline was proportionately increased (p less than 0.05). Similar changes of a greater magnitude were seen when comparing myelomeningoceles to controls. In both types of scoliosis, acid phosphatase was elevated in nuclear and annular tissue. Acid phosphatase activity and hexosamine varied inversely in the nucleus. Finding similar biochemical patterns in idiopathic and neurovascular scoliosis raises the possibility that these changes may be secondary.

Spinal muscle in scoliosis. Part 1. Histology and histochemistry.

Histological and histochemical techniques were employed in a study of the spinal muscles from 35 adolescent scoliotic patients. Many abnormalities were found and, in general, the changes were indicative of denervation and neuropathy. None of the abnormalities were found at any one site more frequently than at any other, nor were they more often seen on one side or the other. None of the patients examined were normal at all sites of biopsy. These findings provide evidence of a neuropathic process being involved in the aetiology or progression of scoliosis. So-called "idiopathic" scoliosis may, perhaps, be the result of an otherwise sub-clinical lower motor neurone disorder.

Amino-oxidase and dermal connective tissue in scoliosis.

The amino-oxidase and collagen content of dermal tissue was evaluated in 10 patients with severe idiopathic scoliosis and in a control group of 7 patients with poliomyelitic scoliosis, that were to undergo surgical correction of their skeletal deformity. A greater dermal content of neutral salt souble collagen was found in the patients with idiopathic scoliosis, compared with the control group. Statistically the difference was highly significant. Mono-amino-oxidase activity, however, was found to be significantly decreased in dermal extracts obtained from patients affected by idiopathic scoliosis in comparison with controls. The involvement of mono-amino-oxidase in the crosslinking of collagen and the possible classification of idiopathic scoliosis as a systemic disease of connective tissue are discussed.