Collagen-related diseases

胶原蛋白相关疾病

• 批准号: 10915309
• 负责人: Sergey Leikin
• 金额: $ 164.54万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10915309
• 关键词: Affect; Aging; Amniotic Fluid; Anabolism; Animals; Arginine; Autophagocytosis; Birth; Breathing; Cell Culture Techniques; Cell Line; Cells; Cellular Stress; Cellular biology; Clinical; Clinical Research; Clustered Regularly Interspaced Short Palindromic Repeats; Coat Protein Complex I; Collaborations; Collagen; Collagen Type I; Complex; Connective Tissue; Connective Tissue Diseases; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Deformity; Degenerative polyarthritis; Deposition; Development; Diagnostic; Disease; Dysplasia; Ehlers-Danlos Syndrome; Endoplasmic Reticulum; Enzymes; Epiphysial cartilage; Evaluation; Event; Extracellular Matrix; Extramural Activities; FLP recombinase; Failure; Fibroblasts; Fibrosis; Fracture; Genes; Glycine; Goals; Golgi Apparatus; Growth; Heat-Shock Proteins 70; Heterozygote; Human; Hydroxylation; Image; In Situ; Knock-out; Knowledge; Ligaments; Longevity; Lung; Lysosomes; Malignant Neoplasms; McCune-Albright Syndrome; Mitochondria; Modeling; Molecular; Movement; Mus; Mutation; Newborn Infant; Osteoblasts; Osteogenesis Imperfecta; Osteoporosis; Pathogenicity; Pathology; Pathway interactions; Patients; Perinatal mortality demographics; Positioning Attribute; Procollagen; Procollagen-Proline Dioxygenase; Proline; Protein Subunits; Proteins; Pulmonary Pathology; Quality Control; Recycling; Research Personnel; Resources; Scientist; Signal Transduction; Site; Skin; Stress; Structure; Technology; Tendon structure; Testing; Tissues; Translations; United States National Institutes of Health; Uterine Fibroids; Uterus; Zebrafish; biological adaptation to stress; bone; bone fragility; endoplasmic reticulum stress; fetal; improved; in vivo; interest; live cell imaging; lung failure; molecular pathology; mortality; mouse model; mutant; new therapeutic target; novel; prevent; recruit; response; rib bone structure; scaffold; sensor; skeletal; skeletal abnormality; skeletal dysplasia; targeted treatment; therapeutic target; trafficking; transcriptome sequencing; translational study; triple helix; vesicle transport

Type I collagen (Col-I) is the most abundant human protein forming the structural scaffold (matrix) of bone, skin and other tissues. Among prominent pathologies associated with disruptions in Col-I biosynthesis are bone fragility and lung failure in OI as well as skin, tendon, and ligament laxity in EDS. Over 80% of severe OI cases are caused by substitutions of glycine (Gly) required in every third position of collagen triple helix, the main functional unit of Col-I. By altering the helix folding, Gly substitutions cause malfunction of bone producing cells (osteoblasts) and alter formation and function of the collagen matrix in tissues. Our studies of mouse models and cells from OI patients revealed both effects to be pathogenic. We demonstrated that a key factor in bone failure is cell stress and resulting osteoblast malfunction due to Endoplasmic Reticulum (ER) disruption by mutant procollagen (collagen precursor) misfolding. We described noncanonical features of this stress, but the mechanism of its activation remained unknown. Recently, we identified ER-mitochondria contacts as the likely sensor of the ER disruption in an OI mouse model, suggesting a novel stress mechanism and novel therapeutic targets. We also demonstrated that lung pathology in the same mice is caused by deficient formation and function of collagen matrix incorporating secreted mutant molecules. To understand and target these pathologies, we created and characterized a G610C mouse model of OI, which mimics a Gly610 to Cys substitution in the alpha-2 chain of Col-I in a large group of patients. This model was instrumental in demonstrating that Gly substitutions may disrupt the osteoblast ER and trigger integrated stress response of the cell without activating canonical ER stress pathways. We then utilized single-cell and spatially resolved in-situ RNA sequencing to reveal activation of mitochondrial stress response via disruption of ER-mitochondria contacts. We showed this response to be regulated by mitochondrial HSP70 and ATF5, which are paralogues of BIP (ER HSP70) and ATF4 regulating canonical ER stress. After confirming this novel response to ER disruption, we are developing a new mouse model with the G610C mutation and ATF5 knockout to investigate the underlying molecular events and find potential therapeutic targets. While bone fragility and skeletal deformities due to osteoblast malfunction are the most discussed pathologies in OI, lung failure is the main cause of mortality in OI, particularly in newborns. Since we observed perinatal lethality of all homozygous and few heterozygous G610C animals due to lung failure, we initiated studies of molecular mechanisms underlying this pathology. In contrast to bones, we found lung pathology to be caused by mutant collagen affecting extracellular matrix and response of lung cells to this matrix rather to be caused by mutant collagen disrupting ER of lung cells. By combining structural characterization of the matrix with single-cell and spatially resolved RNA sequencing, we identified a previously unrecognized mechanism of lung pathology, which likely affects not only OI but many other skeltal dysplasia patients. Specifically, rib cage fractures and deformities as well as deficiency and weakness of the lung collagen matrix prevent proper lung inflation with amniotic fluid during fetal breathing movements, which leads to lung hypoplasia. The recognition of likely lung hypoplasia at birth, its evaluation, and timely treatment may save many lives. Overall, our translational studies include collaborations with many intramural and extramural scientists and clinicians on mechanisms of pathology in OI and other collagen-related disorders. Over the years, we assisted Dr. Marini in discovering novel forms of OI and characterizing underlying pathology. In collaboration with Dr. Byers, we investigated OI caused by arginine substitutions in type I collagen, demonstrating features similar to Gly substitutions. We assisted Dr. Bonnemann in characterization of a complex connective tissue disorder involving pathology of multiple tissues due to deficient function of prolyl 4 hydroxylase 1, an enzyme hydroxylating proline in Col-I. In collaboration with Dr. Stratakis, we described abnormal maturation and function of osteoblasts caused by deficiencies in catalytic and regulatory subunits of protein kinase A that disrupt cAMP signaling, which was reminiscent of McCune-Albright syndrome (constitutively overactive cAMP signaling). In collaboration with Dr. Leppert, we described abnormal composition of collagen deposited in uterine fibromas, which could be involved in the dysregulation of uterine fibroblasts underlying this pathology. We are currently collaborating with Dr. Otsuru on studies of growth plate pathology and growth deficiency in G610C mice. We are also collaborating with Dr. Forlino on characterization of type I collagen processing in zebra fish models of OI. We are also pursuing more fundamental cell biology of procollagen biosynthesis by osteoblasts and fibroblasts, the goal of which is understanding presently unknown molecular mechanisms for subsequent translation into clinical research. For instance, observations made by us and others suggest that disruptions in secretory trafficking and degradation of procollagen might be involved in a many pathologies spanning the entire lifespan, from skeletal dysplasia in early development to osteoporosis in aging. Better understanding procollagen trafficking and degradation might therefore reveal new therapeutic targets and approaches. To study these mechanisms, we developed novel fluorescent constructs of procollagen for live cell imaging. Contrary to popular models, we found procollagen to be delivered to Golgi from ER exit sites (ERESs) by rapidly moving transport vesicles that have no COPII coat and that are dependent on COPI coat formation. We also discovered that misfolded procollagen is recognized at ERES and rerouted from the secretory pathway to a novel autophagy (lysosomal degradation) pathway we termed ERES micro-autophagy, in which ERESs containing misfolded molecules are directly engulfed by lysosomes. We are investigating the mechanism of the lysosomal recruitment to ERES as a potential target for therapeutic applications. We are also investigating whether ERES micro-autophagy is a general quality control mechanism utilized by cells for many proteins and not just procollagen. To facilitate these studies, we have recently utilized CRISPR/CAS gene editing technology for creating several osteoblast cell lines, in which endogenous procollagen is fluorescently tagged and can be manipulated by Flp-recombinase to introduce mutations and change the fluorescent tags. We are using these cells to confirm and expand our knowledge of procollagen trafficking and autophagy. We are also providing them to researchers outside NIH as a useful resource for their studies of collagen-related pathologies. Together, our translational and fundamental studies suggest that autophagy of misfolded procollagen is a crucial adaptation mechanism in OI. To test this hypothesis, we created an OI mouse model for autophagy manipulation by altering Atg5 expression. Using this model, we discovered that osteoblasts recycle misfolded procollagen primarily by ERES micro-autophagy not just in cell culture but also in vivo. We also observed that 3-4-fold reduction in Atg5 increased perinatal lethality of heterozygous G610C mice due to lung failure from < 10% to 50%. We hypothesize that deficient overall animal adaptation to the reduced Atg5 and its effects on the fetal breathing movements cause the increased lethality. Understanding the underlying adaptation mechanisms may help finding new treatment targets for improving the survival of OI babies and minimizing long-term lung pathology in OI.

I型胶原（Col-I）是最丰富的人类蛋白质，形成骨、皮肤和其他组织的结构支架（基质）。与col - 1生物合成中断相关的主要病理包括成骨不全患者的骨脆性和肺衰竭，以及EDS患者的皮肤、肌腱和韧带松弛。超过80%的严重成骨不全病例是由于coli的主要功能单位胶原三螺旋每三分之一的位置需要替换甘氨酸（Gly）引起的。通过改变螺旋折叠，Gly取代引起骨生成细胞（成骨细胞）的功能障碍，并改变组织中胶原基质的形成和功能。我们对小鼠模型和成骨不全患者细胞的研究表明，这两种效应都具有致病性。我们证明了骨衰竭的一个关键因素是细胞应激和由于突变前胶原（胶原前体）错误折叠导致内质网（ER）破坏而导致的成骨细胞功能障碍。我们描述了这种应激的非规范特征，但其激活机制仍然未知。最近，我们在OI小鼠模型中发现内质网线粒体接触可能是内质网破坏的传感器，这提示了一种新的应激机制和新的治疗靶点。我们还证明，同一小鼠的肺部病理是由含有分泌突变分子的胶原基质的形成和功能缺陷引起的。

项目成果

Celecoxib treatment of fibrous dysplasia (FD) in a human FD cell line and FD-like lesions in mice with protein kinase A (PKA) defects.

• DOI: 10.1016/j.mce.2016.08.004
• 发表时间: 2017-01-05
• 期刊: MOLECULAR AND CELLULAR ENDOCRINOLOGY
• 影响因子: 4.1
• 作者: Saloustros, Emmanouil;Liu, Sisi;Mertz, Edward L.;Bhattacharyya, Nisan;Starost, Matthew F.;Salpea, Paraskevi;Nesterova, Maria;Collins, Michael;Leikin, Sergey;Stratakis, Constantine A.
• 通讯作者: Stratakis, Constantine A.

The chaperone activity of 4PBA ameliorates the skeletal phenotype of Chihuahua, a zebrafish model for dominant osteogenesis imperfecta.

• DOI: 10.1093/hmg/ddx171
• 发表时间: 2017-08-01
• 期刊: Human molecular genetics
• 影响因子: 3.5
• 作者: Gioia R;Tonelli F;Ceppi I;Biggiogera M;Leikin S;Fisher S;Tenedini E;Yorgan TA;Schinke T;Tian K;Schwartz JM;Forte F;Wagener R;Villani S;Rossi A;Forlino A
• 通讯作者: Forlino A

Chaperoning osteogenesis: new protein-folding disease paradigms.

• DOI: 10.1016/j.tcb.2010.11.007
• 发表时间: 2011-03
• 期刊: Trends in cell biology
• 影响因子: 19
• 作者: Makareeva E;Aviles NA;Leikin S
• 通讯作者: Leikin S

Zebrafish Collagen Type I: Molecular and Biochemical Characterization of the Major Structural Protein in Bone and Skin.

• DOI: 10.1038/srep21540
• 发表时间: 2016-02-15
• 期刊: Scientific reports
• 影响因子: 4.6
• 作者: Gistelinck C;Gioia R;Gagliardi A;Tonelli F;Marchese L;Bianchi L;Landi C;Bini L;Huysseune A;Witten PE;Staes A;Gevaert K;De Rocker N;Menten B;Malfait F;Leikin S;Carra S;Tenni R;Rossi A;De Paepe A;Coucke P;Willaert A;Forlino A
• 通讯作者: Forlino A

Haploinsufficiency for either one of the type-II regulatory subunits of protein kinase A improves the bone phenotype of Prkar1a+/- mice.

蛋白激酶 A 的任一 II 型调节亚基的单倍体不足可改善 Prkar1a /- 小鼠的骨表型。

• DOI: 10.1093/hmg/ddv320
• 发表时间: 2015
• 期刊: Human molecular genetics
• 影响因子: 3.5
• 作者: Liu,Sisi;Saloustros,Emmanouil;Mertz,EdwardL;Tsang,Kitman;Starost,MatthewF;Salpea,Paraskevi;Faucz,FabioR;Szarek,Eva;Nesterova,Maria;Leikin,Sergey;Stratakis,ConstantineA
• 通讯作者: Stratakis,ConstantineA