Dysregulation of TNF-induced protein 3 and CCAAT/enhancer-binding protein ß in alveolar macrophages: Implications for systemic sclerosis-associated interstitial lung disease.

OBJECTIVES: This study investigates the role of TNF-induced protein 3 (TNFAIP3) and CCAAT/enhancer-binding protein ß (C/EBPß) in alveolar macrophages (AMs) of patients with systemic sclerosis-associated interstitial lung disease (SSc-ILD) and their influence on pulmonary fibrosis. METHODS: Transfection of HEK293T cells and AMs with plasmids carrying TNFAIP3 and C/EBPß was performed, followed by co-culturing AMs with pulmonary fibroblasts. Immunoblotting analysis was then utilized to assess the expression of TNFAIP3, C/EBPß, and collagen type 1 (Col1). Quantitative PCR analysis was conducted to quantify the mRNA levels of C/EBPß, IL-10, and TGF-ß1. STRING database analysis, and immunoprecipitation assays were employed to investigate the interactions between TNFAIP3 and C/EBPß. RESULTS: TNFAIP3 expression was significantly reduced in SSc-ILD AMs, correlating with increased Col1 production in fibroblasts. Overexpression of TNFAIP3 inhibited this pro-fibrotic activity. Conversely, C/EBPß expression was elevated in SSc-ILD AMs, and its reduction through TNFAIP3 restoration decreased pro-fibrotic cytokines IL-10 and TGFß1 levels. Protein-protein interaction studies confirmed the regulatory relationship between TNFAIP3 and C/EBPß. CONCLUSIONS: This study highlights the important role of TNFAIP3 in regulating pulmonary fibrosis in SSc-ILD by modulating C/EBPß expression in AMs. These findings suggest that targeting TNFAIP3 could be a potential therapeutic strategy for managing SSc-ILD patients.

Delivery of miR-29a improves the permeability of cisplatin by downregulating collagen I expression.

In the clinical setting, chemotherapy is the most widely used antitumor treatment, however, chemotherapy resistance significantly limits its efficacy. Reduced drug influx is a key mechanism of chemoresistance, and inhibition of the complexity of the tumor microenvironment (TME) may improve chemotherapy drug influx and therapeutic efficiency. In the current study, we identified that the major extracellular matrix protein collagen I is more highly expressed in lung cancer tissues than adjacent tissues in patients with lung cancer. Furthermore, Kaplan-Meier analysis suggested that COL1A1 expression was negatively correlated with the survival time of patients with lung cancer. Our previous study demonstrated that miR-29a inhibited collagen I expression in lung fibroblasts. Here, we investigated the effect of miR-29a on collagen I expression and the cellular behavior of lung cancer cells. Our results suggest that transfection with miR-29a could prevent Lewis lung carcinoma (LLC) migration by downregulating collagen I expression, but did not affect the proliferation, apoptosis, and cell cycle of LLC cells. In a 3D tumoroid model, we demonstrated that miR-29a transfection significantly increased cisplatin (CDDP) permeation and CDDP-induced cell death. Furthermore, neutral lipid emulsion-based miR-29a delivery improved the therapeutic effect of cisplatin in an LLC spontaneous tumor model in vivo. In summary, this study shows that targeting collagen I expression in the TME contributes to chemotherapy drug influx and improves therapeutic efficacy in lung cancer.

RNA-based bone histomorphometry: method and its application to explaining postpubertal bone gain in a G610C mouse model of osteogenesis imperfecta.

Bone histomorphometry is a well-established approach to assessing skeletal pathology, providing a standard evaluation of the cellular components, architecture, mineralization, and growth of bone tissue. However, it depends in part on the subjective interpretation of cellular morphology by an expert, which introduces bias. In addition, diseases like osteogenesis imperfecta (OI) and fibrous dysplasia are accompanied by changes in the morphology and function of skeletal tissue and cells, hindering consistent evaluation of some morphometric parameters and interpretation of the results. For instance, traditional histomorphometry combined with collagen turnover markers suggested that reduced bone formation in classical OI is accompanied by increased bone resorption. In contrast, the well-documented postpubertal reduction in fractures would be easier to explain by reduced bone resorption after puberty, highlighting the need for less ambiguous measurements. Here we propose an approach to histomorphometry based on in situ mRNA hybridization, which uses Col1a1 as osteoblast and Ctsk as osteoclast markers. This approach can be fully automated and eliminates subjective identification of bone surface cells. We validate these markers based on the expression of Bglap, Ibsp, and Acp5. Comparison with traditional histological and tartrate-resistant acid phosphatase staining of the same sections suggests that mRNA-based analysis is more reliable. Unlike inconclusive traditional histomorphometry of mice with α2(I)-Gly610 to Cys substitution in the collagen triple helix, mRNA-based measurements reveal reduced osteoclastogenesis in 11-wk-old animals consistent with the postpubertal catch-up osteogenesis observed by microCT. We optimize the technique for cryosections of mineralized bone and sections of paraffin-embedded decalcified tissue, simplifying and broadening its applications. We illustrate the application of the mRNA-based approach to human samples using the example of a McCune-Albright syndrome patient. By eliminating confounding effects of altered cellular morphology and the need for subjective morphological evaluation, this approach may provide a more reproducible and accessible evaluation of bone pathology.

The association between Dental Fluorosis and COL1A2 gene polymorphism among a Tunisian Population.

Dental fluorosis (DF) is a prevalent developmental defect of tooth enamel caused by exposure to excessive fluoride, with the severity dependent on various factors. This study aimed to investigate the association between DF and a specific genetic polymorphism (rs412777) in the COL1A2 gene among a Tunisian population. A case-control study was conducted from July to November 2022, involving a total of 95 participants including 51 cases and 44 controls. Dental examinations and genetic analysis were performed to assess the relationship between the COL1A2 gene polymorphism and DF.The results of allelic distribution revealed that A allele carriers were significantly protected against (DF) when compared to those with the C allele (C vs. A, p = 0.001; OR = 0.375 (0.207-0.672)). This suggests a strong correlation between the presence of the C allele and the risk of developing DF. Additionally, significant association between the CC genotype of rs412777 and an increased risk of DF was found under both codominant and dominant genetic models (P = 0.002 and P < 0.001 respectively).The findings suggest that genetic predisposition plays a relevant role in the development of DF. Further research is needed to explore the potential use of genetic markers for DF and their implications for public health. This study provides the first insights into the genetic factors associated with DF in the Tunisian population, contributing to our understanding of this prevalent dental condition.

Type 1 collagen: Synthesis, structure and key functions in bone mineralization.

Collagen is a highly abundant protein in the extracellular matrix of humans and mammals, and it plays a critical role in maintaining the body's structural integrity. Type I collagen is the most prevalent collagen type and is essential for the structural integrity of various tissues. It is present in nearly all connective tissues and is the main constituent of the interstitial matrix. Mutations that affect collagen fiber formation, structure, and function can result in various bone pathologies, underscoring the significance of collagen in sustaining healthy bone tissue. Studies on type 1 collagen have revealed that mutations in its encoding gene can lead to diverse bone diseases, such as osteogenesis imperfecta, a disorder characterized by fragile bones that are susceptible to fractures. Knowledge of collagen's molecular structure, synthesis, assembly, and breakdown is vital for comprehending embryonic and foetal development and several aspects of human physiology. In this review, we summarize the structure, molecular biology of type 1 collagen, its biomineralization and pathologies affecting bone.

Effect of Vaginal Microecological Alterations on Female Pelvic Organ Prolapse.

INTRODUCTION AND HYPOTHESIS: The objective was to investigate the correlation between endogenous vaginal microecological alterations and female pelvic organ prolapse (POP). METHODS: Patients who underwent vaginal hysterectomy were retrospectively analyzed as the POP group (n = 30) and the non-POP group (n = 30). The vaginal microbial metabolites and enzyme levels were tested using the dry chemoenzymatic method. The mRNA and protein expression were tested using real-time quantitative PCR and immunohistochemistry. SPSS version 25.0 and GraphPad Prism 8.0 were performed for statistical analysis. RESULTS: Compared with the non-POP group, the vaginal pH, H2O2 positivity and leukocyte esterase positivity were higher in patients with POP (all p < 0.05). Further analysis showed that patients with pelvic organ prolapse quantification (POP-Q) stage IV had higher rates of vaginal pH, H2O2 positivity and leukocyte esterase positivity than those with POP-Q stage III. Additionally, the mRNA expression of decorin (DCN), transforming growth factor beta 1 (TGF-ß1), and matrix metalloproteinase-3 (MMP-3) in uterosacral ligament tissues were higher, whereas collagen I and III were lower. Similarly, the positive expression of MMP-3 in uterosacral ligament tissue was significantly upregulated in the POP group compared with the non-POP group (p = 0.035), whereas collagen I (p = 0.004) and collagen III (p = 0.019) in uterosacral ligament tissue were significantly downregulated in the POP group. Correlation analysis revealed that there was a significant correlation between vaginal microecology and collagen metabolism. In addition, MMP-3 correlated negatively with collagen I and collagen III (p = 0.002, r = -0.533; p = 0.002, r = -0.534 respectively), whereas collagen I correlated positively with collagen III (p = 0.001, r = 0.578). CONCLUSIONS: Vaginal microecological dysbiosis affects the occurrence of female POP, which could be considered a novel therapeutic option.

Echocardiographic abnormalities and joint hypermobility in Chinese patients with Osteogenesis imperfecta.

BACKGROUND: Very little is known about the characteristics of echocardiographic abnormalities and joint hypermobility in Chinese patients with osteogenesis imperfecta (OI). The aim of our study was to investigate the characteristics, prevalence and correlation of echocardiographic abnormalities and joint hypermobility in Chinese patients with OI. METHODS: A cross-sectional comparative study was conducted in pediatric and adult OI patients who were matched in age and sex with healthy controls. Transthoracic echocardiography was performed in all patients and controls, and parameters were indexed for body surface area (BSA). The Beighton score was used to evaluate the degree of joint hypermobility. RESULTS: A total of 48 patients with OI (25 juveniles and 23 adults) and 129 age- and sex-matched healthy controls (79 juveniles and 50 adults) were studied. Four genes (COL1A1, COL1A2, IFITM5, and WNT1) and 39 different mutation loci were identified in our study. Mild valvular regurgitation was the most common cardiac abnormality: mild mitral and tricuspid regurgitation was found in 12% and 36% of pediatric OI patients, respectively; among 23 OI adults, 13% and 17% of patients had mild mitral and tricuspid regurgitation, respectively, and 4% had mild aortic regurgitation. In multiple regression analysis, OI was the key predictor of left atrium diameter (LAD) (ß=-3.670, P < 0.001) and fractional shortening (FS) (ß = 3.005, P = 0.037) in juveniles, whereas for adults, OI was a significant predictor of LAD (ß=-3.621, P < 0.001) and left ventricular mass (LVM) (ß = 58.928, P < 0.001). The percentages of generalized joint hypermobility in OI juveniles and adults were 56% and 20%, respectively. Additionally, only in the OI juvenile group did the results of the Mann‒Whitney U test show that the degree of joint hypermobility was significantly different between the echocardiographic normal and abnormal groups (P = 0.004). CONCLUSIONS: Mild valvular regurgitation was the most common cardiac abnormality in both OI juveniles and adults. Compared with OI adults, OI juveniles had more prevalent and wider joint hypermobility. Echocardiographic abnormalities may imply that the impairment of type I collagen is more serious in OI. Baseline echocardiography should be performed in OI patients as early as possible.

Exome sequencing identified mutations in the WNT1 and COL1A2 genes in osteogenesis imperfecta cases.

BACKGROUND: Osteogenesis imperfecta (OI) is a heritable connective tissue disorder characterized by bone deformities, fractures and reduced bone mass. OI can be inherited as a dominant, recessive, or X-linked disorder. The mutational spectrum has shown that autosomal dominant mutations in the type I collagen-encoding genes are responsible for OI in 85% of the cases. Apart from collagen genes, mutations in more than 20 other genes, such as CRTAP, CREB3L1, MBTPS2, P4HB, SEC24D, SPARC, FKBP10, LEPRE1, PLOD2, PPIB, SERPINF1, SERPINH1, SP7, WNT1, BMP1, TMEM38B, and IFITM5 have been reported in OI. METHODS AND RESULTS: To understand the genetic cause of OI in four cases, we conducted whole exome sequencing, followed by Sanger sequencing. In case #1, we identified a novel c.506delG homozygous mutation in the WNT1 gene, resulting in a frameshift and early truncation of the protein at the 197th amino acid. In cases #2, 3 and 4, we identified a heterozygous c.838G > A mutation in the COL1A2 gene, resulting in a p.Gly280Ser substitution. The clinvar frequency of this mutation is 0.000008 (GnomAD-exomes). This mutation has been identified by other studies as well and appears to be a mutational hot spot. These pathogenic mutations were found to be absent in 96 control samples analyzed for these sites. The presence of these mutations in the cases, their absence in controls, their absence or very low frequency in general population, and their evaluation using various in silico prediction tools suggested their pathogenic nature. CONCLUSIONS: Mutations in the WNT1 and COL1A2 genes explain these cases of osteogenesis imperfecta.

Effects of pulsed electrical stimulation on α-smooth muscle actin and type I collagen expression in human dermal fibroblasts.

Pulsed electrical stimulation (PES) is known to affect cellular activities. We previously found PES to human dermal fibroblasts (HFs) promoted platelet-derived growth factor subunit A (PDGFA) gene expression, which enhanced proliferation. In this study, we investigated PES effects on fibroblast collagen production and differentiation into myofibroblasts. HFs were electrically stimulated at 4800 Hz and 5 V for 60 min. Imatinib, a specific inhibitor of PDGF receptors, was treated before PES. After 6 h of PES, PDGFA, α-smooth muscle actin (α-SMA), and collagen type I α1 chain gene expressions were upregulated in PES group. Imatinib suppressed the promoted expression except for PDGFA. Immunofluorescence staining and enzyme-linked immunosorbent assay showed the production of α-SMA and collagen I was enhanced in PES group but suppressed in PES + imatinib group at 48 h after PES. Therefore, PES promotes the production of α-SMA and collagen I in fibroblasts, which is triggered by PDGFA that is upregulated early after PES.

Identification of a family with van der Hoeve's syndrome harboring a novel COL1A1 mutation and generation of patient-derived iPSC lines and CRISPR/Cas9-corrected isogenic iPSCs.

Van der Hoeve's syndrome, also known as osteogenesis imperfecta (OI), is a genetic connective tissue disorder characterized by fragile, fracture-prone bone and hearing loss. The disease is caused by a gene mutation in one of the two type I collagen genes COL1A1 or COL1A2. In this study, we identified a novel frameshift mutation of the COL1A1 gene (c.1607delG) in a family with OI using whole-exome sequencing, bioinformatics analysis and Sanger sequencing. This mutation may lead to the deletion of a portion of exon 23 and the generation of a premature stop codon in the COL1A1 gene. To further investigate the impact of this mutation, we established two induced pluripotent stem cell (iPSC) lines from peripheral blood mononuclear cells of OI patients carrying a novel mutation in the COL1A1 gene. Osteoblasts (OB) derived from OI-iPSCs exhibited reduced production of type I collagen and diminished ability to differentiate into osteoblasts. Using a CRISPR-based homology-directed repair strategy, we corrected the OI disease-causing COL1A1 novel mutations in iPSCs generated from an affected individual. Our results demonstrated that the diminished expression of type I collagen and osteogenic potential were enhanced in OB induced from corrected OI-iPSCs compared to those from OI-iPSCs. Overall, our results provide new insights into the genetic basis of Van der Hoeve's syndrome and highlight the potential of iPSC technology for disease modeling and therapeutic development.

1-Kestose Blocks UVB-Induced Skin Inflammation and Promotes Type I Procollagen Synthesis via Regulating MAPK/AP-1, NF-κB and TGF-ß/Smad Pathway.

Solar UVB irradiation cause skin photoaging by inducing the high expression of matrix metalloproteinase (MMPs) to inhibit the expression of Type1 procollagen synthesis. 1-Kestose, a natural trisaccharide, has been indicated to show a cytoprotective role in UVB radiation-induced-HaCaT cells. However, few studies have confirmed the anti-aging effects. In the present study, we evaluated the anti-photoaging and pathological mechanism of 1-kestose using Human keratinocytes (HaCaT) cells. The results found that 1-kestose pretreatment remarkably reduced UVB-generated reactive oxygen species (ROS) accumulation in HaCaT cells. 1-Kestose suppressed UVB radiation-induced MMPs expressions by blocking MAPK/AP-1 and NF-κB p65 translocation. 1-Kestose pretreatment increased Type 1 procollagen gene expression levels by activating TGF-ß/Smad signaling pathway. Taken together, our results demonstrate that 1-kestose may serve as a potent natural trisaccharide for inflammation and photoaging prevention.

Study on the molecular mechanism of dihydromyricetin in alleviating liver cirrhosis based on network pharmacology.

Dihydromyricetin (DHM) is a bioactive flavonoid extracted from Hovenia dulcis, which has various activities. In the present study, the molecular mechanism of dihydromyricetin (DHM) in relieving liver cirrhosis was investigated through network pharmacology and experimental verification. The cell model was induced by TGF-ß1 activating the human hepatic stellate cell line (HSC; LX-2). The protein levels of α-SMA, collagen I, and collagen III and pathway-related proteins within LX-2 cells were detected using Western blot. EdU staining was conducted to detect cell proliferation. Immunofluorescence staining was performed to detect the expression levels of α-SMA and collagen I. Next, the drug targets of DHM were screened from the PubChem database. The differentially expressed genes in the liver cirrhosis dataset GSE14323 were identified. The expression of the identified drug targets in LX-2 cells was verified using qRT-PCR. The results showed that TGF-ß1 treatment notably increased LX-2 cell viability, promoted cell proliferation, and elevated α-SMA, collagen I, and collagen III protein contents. DHM treatment could partially eliminate TGF-ß1 effects, as evidenced by the inhibited cell viability and proliferation and reduced α-SMA, collagen I, and collagen III contents. After network pharmacology analysis, nine differentially expressed target genes (MMP2, PDGFRB, PARP1, BCL2L2, ABCB1, TYR, CYP2E1, SQSTM1, and IL6) in liver cirrhosis were identified. According to qRT-PCR verification, DHM could inhibit the expression of MMP2, PDGFRB, PARP1, CYP2E1, SQSTM1, and IL6, and enhance ABCB1 expression levels within LX-2 cells. Moreover, DHM inhibited mTOR and MAPK signaling pathways in TGF-ß1-induced HSCs. In conclusion, DHM could inhibit HSC activation, which may be achieved via acting on MMP2, PDGFRB, PARP1, CYP2E1, SQSTM1, IL6, and ABCB1 genes and their downstream signaling pathways, including mTOR and MAPK signaling pathway.

IL32 downregulation lowers triglycerides and type I collagen in di-lineage human primary liver organoids.

Steatotic liver disease (SLD) prevails as the most common chronic liver disease yet lack approved treatments due to incomplete understanding of pathogenesis. Recently, elevated hepatic and circulating interleukin 32 (IL-32) levels were found in individuals with severe SLD. However, the mechanistic link between IL-32 and intracellular triglyceride metabolism remains to be elucidated. We demonstrate in vitro that incubation with IL-32ß protein leads to an increase in intracellular triglyceride synthesis, while downregulation of IL32 by small interfering RNA leads to lower triglyceride synthesis and secretion in organoids from human primary hepatocytes. This reduction requires the upregulation of Phospholipase A2 group IIA (PLA2G2A). Furthermore, downregulation of IL32 results in lower intracellular type I collagen levels in di-lineage human primary hepatic organoids. Finally, we identify a genetic variant of IL32 (rs76580947) associated with lower circulating IL-32 and protection against SLD measured by non-invasive tests. These data suggest that IL32 downregulation may be beneficial against SLD.

Comprehensive transcriptional analysis of early dorsal skin development in pigs.

Porcine skin is similar to human skin in physiology, anatomy and histology and is often used as a model animal for human skin research. There are few studies on the transcriptome aspects of pig skin during the embryonic period. In this study, RNA sequencing was performed on the dorsal skin of Chenghua sows at embryonic day 56 (E56), embryonic day 76 (E76), embryonic day 105 (E105), and 3 days after birth (D3) to explore RNA changes in pig dorsal skin at four ages. A number of skin-related differential genes were identified by intercomparison between RNAs at four time points, and KEGG functional analysis showed that these differential genes were mainly enriched in metabolic and developmental, immune, and disease pathways, and the pathways enriched in GO analysis were highly overlapping. Collagen is an important part of the skin, with type I collagen making up the largest portion. In this study, collagen type I alpha 1 (COL1A1) and collagen type I alpha 2 (COL1A2) were significantly upregulated at four time points. In addition, lncRNA-miRNA-mRNA and miRNA-circRNA coexpression networks were constructed. The data obtained may help to explain age-related changes in transcriptional patterns during skin development and provide further references for understanding human skin development at the molecular level.

Dietary rice bran attenuates hepatic stellate cell activation and liver fibrosis in mice through enhancing antioxidant ability.

Various endogenous and exogenous stimuli can result in an inflammatory response and collagen deposition in the liver, which affect liver function and increase the risk of developing liver cirrhosis and cancer. Rice bran, the main by-product of rice milling, contains various nutrients which possess hepatoprotective activities. In this study, we investigated the effects of rice bran on carbon tetrachloride (CCl4)-induced liver fibrosis in mice. Mice were fed a rice-bran-containing diet (10% rice bran w/w) or a standard diet with or without an injection of 20% CCl4 to induce liver fibrosis. Our results showed that feeding a rice-bran-containing diet could alleviate CCl4-induced liver damage, collagen deposition, and expressions of fibrosis-related genes, including α-smooth muscle actin (α-SMA), collagen 1a2 (COL1A2), and transforming growth factor-ß (TGF-ß) in liver tissues. Moreover, consumption of rice bran enhanced phase II detoxification and antioxidant gene expressions, including Gsta3, Gstp1, Catalase, SOD1, SOD2, and SOD3. Treatment with γ-oryzanol, the major bioactive compound in rice bran, decreased the sensitivity of hepatic stellate cells (HSCs) to TGF-ß1-induced α-SMA, COL1A2, and phosphorylated smad2 expressions. In conclusion, a rice-bran-containing diet may have beneficial effects on liver fibrogenesis through increased antioxidant and detoxification activities. γ-Oryzanol, the major bioactive compound of rice bran, can inhibit activation of HSCs.

Effect of Sparc knockout on the extracellular matrix of mouse periodontal ligament cells.

The periodontal ligament (PDL) is a critical component in maintaining tooth stability. It is composed of cells and an extracellular matrix (ECM), each with unique roles in tissue function and homeostasis. Secreted protein acidic and rich in cysteine (SPARC), a calcium-binding matricellular glycoprotein, plays a crucial role in regulating ECM assembly and turnover, alongside facilitating cellular-ECM interactions. In the present study, mass spectrometry-based proteomics was used to assess the impacts of Sparc-knockout (KO) on PDL-derived cells. Results demonstrated that Sparc-KO significantly reduces ECM production and alters its composition with increased levels of type I collagen. Despite this increase in Sparc-KO, type I collagen was not likely to be effectively integrated into the fibrils due to collagen cross-linking impairment. Furthermore, the pathway and process enrichment analyses suggested that SPARC plays a protective role against ECM degradation by antagonistically interacting with cell-surface collagen receptors. These findings provide detailed insights into the multifaceted role of SPARC in ECM organization, including its impact on ECM production, collagen regulation, and interactions with various cellular compartments. A better understanding of these complex mechanisms is crucial for comprehending the causes of periodontal disease and tissue regeneration, where precise control of ECM organization is necessary.

Trichinella spiralis cathepsin B bound and degraded host's intestinal type I collagen.

Trichinellosis is a zoonotic parasitic disease that poses threats to human health, the meat industry, food safety, and huge financial losses. The critical stage of Trichinella spiralis (T. spiralis) infection is the invasion of intestinal larvae into the host's intestinal epithelial cells (IECs). T. spiralis Cathepsin B (TsCB) specifically interacts with IECs to facilitate the invasion of larvae. This study aims to look at how TsCB affects mouse IECs. TsCB was successfully cloned, expressed, and characterized, demonstrating its natural cysteine protease hydrolysis activity. A total of 140 proteins that interact with rTsCB were identified by GST pull-down combined with LC-MS/MS, including type I collagen, an essential component of the host's intestinal epithelial barrier system and intimately related to intestinal epithelial damage. TsCB transcription and expression levels rise, whereas type I collagen in the host's intestinal mucosa declines when the T. spiralis larvae invaded. Besides, it was discovered that TsCB bound to and degraded type I collagen of the host's intestine. This research can serve as a foundation for clarifying how T. spiralis invades the host's intestinal barrier and might provide information on potential targets for the creation of novel treatments to treat parasite illnesses.

Exome sequencing-aided precise diagnosis of four families with type I Stickler syndrome.

BACKGROUND: Stickler syndrome is a multisystemic disorder characterized by ophthalmological and non-ophthalmological abnormalities, frequently misdiagnosed due to high clinical heterogeneity. Stickler syndrome type I (STL1) is predominantly caused by mutations in the COL2A1 gene. METHODS: Exome sequencing and co-segregation analysis were utilized to scrutinize 35 families with high myopia, and pathogenic mutations were identified. Mutant COL2A1 was overexpressed in cells for mechanistic study. A retrospective genotype-phenotype correlation analysis was further conducted. RESULTS: Two novel pathogenic mutations (c.2895+1G>C and c.3505G>A (p.Val1169Ile)) and two reported mutations (c.1597C>T (p.Arg533*) and c.1693C>T (p.Arg565Cys)) in COL2A1 were identified causing STL1. These mutations are all in the G-X-Y triplet, and c.2895+1G>C contributed to aberrant RNA splicing. COL2A1 mutants tended to form large aggregates in the endoplasmic reticulum (ER) and elevated ER stress. Additionally, mutations c.550G>A (p.Ala184Thr) and c.2806G>A (p.Gly936Ser) in COL2A1 were found in high myopia families, but were likely benign, although c.2806G>A (p.Gly936Ser) is on G-X-Y triplet. Moreover, genotype-phenotype correlation analysis revealed that mutations in exon 2 mainly contribute to retinal detachment, whereas mutations in the collagen alpha-1 chain region of COL2A1 tend to cause non-ophthalmologic symptoms. CONCLUSION: This study broadens the COL2A1 gene mutation spectrum, provides evidence for ER stress caused by pathogenic COL2A1 mutations and highlights the importance of non-ophthalmological examination in clinical diagnosis of high myopia.

Collagen prolyl 4-hydroxylase isoenzymes I and II have sequence specificity towards different X-Pro-Gly triplets.

Collagen biosynthesis requires several co- and post-translational modifications of lysine and proline residues to form structurally and functionally competent collagen molecules. Formation of 4-hydroxyproline (4Hyp) in Y-position prolines of the repetitive -X-Y-Gly- sequences provides thermal stability for the triple-helical collagen molecules. 4Hyp formation is catalyzed by a collagen prolyl 4-hydroxylase (C-P4H) family consisting of three isoenzymes. Here we identify specific roles for the two main C-P4H isoenzymes in collagen hydroxylation by a detailed 4Hyp analysis of type I and IV collagens derived from cell and tissue samples. Loss of C-P4H-I results in underhydroxylation of collagen where the affected prolines are not uniformly distributed, but mainly present in sites where the adjacent X-position amino acid has a positively charged or a polar uncharged side chain. In contrast, loss of C-P4H-II results in underhydroxylation of triplets where the X-position is occupied by a negatively charged amino acid glutamate or aspartate. Hydroxylation of these triplets was found to be important as loss of C-P4H-II alone resulted in reduced collagen melting temperature and altered assembly of collagen fibrils and basement membrane. The observed C-P4H isoenzyme differences in substrate specificity were explained by selective binding of the substrate to the active site resulting in distinct differences in Km and Vmax values. Furthermore, our results clearly show that the substrate proline selection is not dependent on the collagen type, but the main determinant is the X-position amino acid of the -X-Pro-Gly- triplet. Although our data clearly shows the necessity of both C-P4H-I and II for normal prolyl 4-hydroxylation and function of collagens, the mRNA expression of the isoenzymes with various procollagens was, surprisingly, not tightly coordinated, suggesting additional levels of control. In conclusion, this study provides a molecular level explanation for the need of multiple C-P4H isoenzymes to generate collagen molecules capable to assemble into intact extracellular matrix structures.