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National Institutes of Health

Eunice Kennedy Shriver National Institute of Child Health and Human Development

2020 Annual Report of the Division of Intramural Research

Extracellular Matrix Disorders: Molecular Mechanisms and Treatment Targets

Sergey Leikin
  • Sergey Leikin, PhD, Head, Section on Physical Biochemistry
  • Elena N. Makareeva, PhD, Staff Scientist
  • Edward L. Mertz, PhD, Staff Scientist
  • Shakib Omari, PhD, Postdoctoral Fellow
  • Laura Gorrell, BS, Predoctoral Fellow
  • Satoru Otsuru, MD, PhD, Special Volunteer

The extracellular matrix (ECM) is responsible for the structural integrity of tissues and organs as well as for maintaining an optimal environment for cellular function. ECM pathology is involved in a wide variety of disorders, ranging from rare genetic abnormalities of skeletal development (skeletal dysplasias) to such common ailments as osteoporosis, fibrosis, and cancer. Collagens are triple-helical proteins that form the structural scaffolds of the ECM. Their procollagen precursors are assembled and folded from three pro-alpha chains in the endoplasmic reticulum (ER), trafficked through the Golgi apparatus, secreted, and then converted into mature collagen by enzymatic cleavage of propeptides. The most common collagen is type I, which is a heterotrimer of two pro-alpha1(I) chains and one pro-alpha2(I) chain and is by far the most abundant protein in all vertebrates. Type I collagen fibers form the organic scaffold of bone, tendons, ligaments, and the matrix of skin and many other tissues. We focus on translational studies of developmental disorders of the ECM such as osteogenesis imperfecta (OI), Ehlers-Danlos syndrome (EDS), and chondrodysplasias, as well as related ECM pathologies in fibrosis, cancer, and osteoporosis. Our goal is to understand molecular mechanisms and thus identify treatment targets in ECM disorders, particularly those involving abnormal metabolism of type I collagen, and to bring this knowledge to clinical research and practice.

Procollagen folding and its role in ECM disorders

Osteoblasts and fibroblasts produce and secrete the massive amounts of type I procollagen needed to build the skeleton and other tissues. Type I procollagen is one of the most difficult proteins to fold. Its massive production presents a unique challenge for protein quality control and trafficking. We discovered that, above 35°C, the conformation of natively folded human procollagen is less favorable than the unfolded one. To fold procollagen at body temperature, cells use specialized ER chaperones to stabilize the native conformation. Outside the cell, the native conformation is stabilized after procollagen is converted to collagen and incorporated into collagen fibers. Unincorporated molecules denature within several hours of secretion and become susceptible to rapid proteolytic degradation. Up to 10–15% of procollagen is misfolded even under normal conditions, necessitating activation of cell stress–response pathways that are responsible for degradation of misfolded molecules and which force the cell to always function in a high-stress mode. Our findings indicate that one of the key pathophysiological mechanisms of OI and other hereditary type I collagen–metabolism disorders is cell stress caused by excessive procollagen misfolding, inability of the cell to handle the normal load of misfolded procollagen, or both.

While we focus mostly on hereditary disorders affecting bone and other tissue development in children, excessive procollagen misfolding of nonhereditary origin is also likely. Our data suggest that such misfolding should occur upon changes in the ER associated with environmental factors, inflammation, aging, etc. It is likely to contribute to fibrosis, cancer, age-related osteoporosis, and many other common ailments. However, little is known about procollagen misfolding pathophysiology because underlying molecular mechanisms and consequences of excessive misfolding for the cell remain poorly understood.

The most common hereditary cause of procollagen misfolding is a Gly substitution anywhere in the obligatory (Gly-X-Y)n sequence that distinguishes all collagens. Such substitutions in type I collagen are responsible for over 80% of severe OI cases. Similar substitutions in other collagens cause EDS and a variety of other syndromes. Their pathophysiology is one of the key topics of our studies. For instance, our studies on OI patients with over 50 different Gly substitutions revealed several structural regions within the collagen where these mutations might be responsible for distinct OI phenotypes. One such region is the first 85–90 amino acids at the N-terminal end of the triple helix, mutations in which prevent normal N-propeptide cleavage. Incorporation of the uncleaved molecules into collagen fibrils leads to distinct OI/EDS with hyperextensibility and joint laxity.

Cell biology of procollagen misfolding

Our current research focuses on the cell biology of procollagen misfolding. In one approach, we use live-cell imaging to investigate the synthesis, folding, trafficking, and degradation of fluorescently tagged procollagen in osteoblasts. Such imaging of osteoblasts transiently transfected with fluorescent procollagen chains revealed new pathways of sorting and trafficking of normally folded and misfolded procollagen molecules in the cell. As expected, normally folded procollagen is loaded into Golgi-bound transport vesicles at ER exit sites (ERES) that are marked by the coat protein complex II (COPII). Contrary to widely held beliefs, however, the vesicles do not have a COPII coat nor do they contain HSP47, a collagen-specific ER chaperone that preferentially binds to natively folded procollagen to assist in its folding and loading into ERES. Instead, transport vesicle formation appears to be dependent on COPI coat assembly and HSP47 release at distal regions of ERES, potentially explaining unusual features of bone pathology caused by mutations in HSP47 and COPI coat proteins. Misfolded procollagen is retained at ERES, resulting in a COPII–dependent modification of ERES membranes by ubiquitin and autophagic machinery. We discovered that such ERESs are then directly engulfed by lysosomes and degraded, delineating a new ERES micro-autophagy pathway. We are currently investigating whether mutations in the COPII protein SEC24D cause bone pathology by disrupting this pathway.

Rerouting of ERES–loaded cargo from secretion to micro-autophagy may have wide implications. It is likely to be a general rather than collagen-specific phenomenon, considering the known COPII coat involvement in both protein secretion and degradation. The hypothesis is currently under investigation in our and several collaborating laboratories. From clinical and translational perspectives, our findings may explain why patients with mutations in different COPII proteins have different pathologies in the development of bone, cartilage, and other tissues.

To gain all these and additional insights, we are expanding the toolbox for studies of collagen metabolism pathology by exploiting emerging gene-editing technologies. We created an osteoblast cell line in which the endogenous pro-alpha2(I) chain has a fluorescent tag and Flp–recombinase target sites for replacing the tag with other peptides. We demonstrated that the cells produce and mineralize bone-like ECM, enabling us to perform live-cell imaging of endogenous procollagen, confirming its trafficking pathways. Given the interest of other researchers in using such cells for a variety of mechanistic studies, we are currently in the process of depositing several clones for public distribution. We also believe that the same strategy can then be used to generate mouse models and study other proteins.

In another approach, we are investigating the pathophysiology of cell-stress response to procollagen misfolding caused by a Gly610 to Cys substitution in the triple-helical region of pro-alpha2(I). We helped develop a mouse model of OI with this mutation (G610C mouse), which mimics the pathology found in a large group of patients with the same mutation. Our study of this model revealed misfolding and accumulation of mutant procollagen in the ER of fibroblasts and osteoblasts, resulting in cell stress and malfunction. We are therefore investigating the mechanism this stress and its role in pathology by altering how the cells adapt to it and by examining cell-stress response pathways activated by the mutation. First, we found that reduced autophagic degradation of misfolded procollagen resulting from reduced expression of an essential autophagy protein ATG5 causes about 40% perinatal lethality of the animals, apparently owing to malfunction of lung fibroblasts. Since lung pathology is the most common cause of death in OI patients, we are examining the underlying molecular mechanisms and potential targets for therapeutic intervention. Second, we demonstrated that knockout of ATG5 in mature osteoblasts increased bone pathology, confirming the pathogenicity of misfolded procollagen accumulation in osteoblast ER in vivo and the role of autophagy in reducing such accumulation. Third, our ongoing single-cell RNA studies are demonstrating that the G610C mutation activates a previously unknown, noncanonical cell-stress response: increased transcription of cell-stress transducer genes Atf5 and Hspa9 instead of their homologs Atf4 and Hspa5 that are known to be involved in unfolded protein response pathways of canonical ER stress. We confirmed this noncanonical cell-stress response by in situ RNA hybridization, identified several candidate pathways involved, and are currently examining the underlying molecular mechanisms.

New approaches to analysis and treatment of ECM pathology

Our observations suggested that the pathology associated with procollagen misfolding may be partially reversed by improving cell adaptation to misfolded procollagen accumulation in the ER, thereby improving lung fibroblast and osteoblast function. Although this would not eliminate the detrimental effects of secreted mutant collagen, pharmacological treatment of cell malfunction is the most realistic short-term strategy for targeting the causes rather than the effect of bone pathology in OI. It is also likely to be a good long-term strategy for the treatment of cell malfunction caused by procollagen misfolding in cases that do not involve pathogenic mutations.

To pursue the strategy, we are examining the effects of enhancing the natural ability of cells to remove and degrade misfolded molecules via autophagy, which is the simplest way to prevent their pathogenic accumulation in the ER. Our preliminary study of autophagy enhancement by a low-protein diet (LPD) in G610C mice revealed improved osteoblast differentiation and function, resulting in better bone quality, but prolonged LPD stunted animal growth. We are thus testing intermittent LPD and fasting approaches that might provide the same benefits of autophagy enhancement without long-term nutrient deficiency.

At the same time, we are validating the general approach of autophagy enhancement by direct genetic modulation of autophagy efficiency. For instance, our studies on the effects of reduced and increased ATG5 expression confirmed that osteoblasts also degrade misfolded procollagen primarily by ERES micro-autophagy in vivo. We found that this autophagy pathway is only moderately affected by ATG5 expression, necessitating a search for other therapeutic targets. We are therefore testing drugs known to reduce the accumulation of misfolded proteins in the ER by enhancing their secretion and autophagy (e.g., 4-phenylbutyrate or 4PBA) and drugs known to reduce the impact of this accumulation on protein synthesis (e.g., ISRIB). We found that 4PBA reduces bone pathology in a zebrafish model of OI and in G610C mice, but that 4PBA is very rapidly metabolized and therefore difficult to deliver to the targeted tissues in the proper therapeutic dosage. Our studies of ISRIB and other drugs are still in exploratory stages.

A key issue in monitoring treatment efficiency in animal models as well as in general diagnostic analysis of bone pathology is the lack of reliable methods with which to characterize the function of bone cells. Traditional histopathology relies on subjective analysis of bone-cell morphology in tissue sections, which is not a reliable indicator of cell function. Over the last two years, we developed a new approach for visualizing and quantifying mRNA expression in individual cells in bone sections. The approach enables objective and reliable cell identification as well as in situ characterization of cell differentiation and function. Based on the interest of bone histomorphometry experts in learning this approach, we hope that it will soon be adapted not only for research but also for clinical practice.

Translational studies on patients with novel or unusual forms of skeletal dysplasia

Over the last several years, we assisted several clinical research groups in characterizing collagen metabolism pathology in cells from patients with newly discovered skeletal dysplasias caused by mutations in cartilage-associated protein (CRTAP), prolyl-3-hydrohylase (P3H1), cyclophilin B (CYPB), the collagen-binding molecular chaperone FKBP65, the signaling protein WNT1, the ER–membrane ion channel TRICB, Golgi-membrane metalloprotease S2P, the transmembrane anterior-posterior transformation protein 1 (TAPT1), or collagen prolyl-4-hydroxylase 1 (P4H1). Our studies suggested that the CRTAP/P3H1/CYPB complex functions as a procollagen chaperone. A deficiency in any of the three proteins delays procollagen folding, although their exact roles remain unclear. More surprisingly, we found no detectable changes in the procollagen folding rate in cultured fibroblasts from patients with FKBP65 mutations. Our data suggested that FKBP65 may affect post-translational modification of procollagen and deposition of collagen matrix by a different mechanism. It remains unclear why some FKBP65 mutations cause severe OI with joint contractures (Bruck's disease), while others cause joint contractures without pronounced OI (Kuskokwim syndrome) or OI without pronounced joint contractures. Our study of TRICB–deficient cells revealed abnormal conformation and reduced thermal stability of type I procollagen, suggesting dysregulation of collagen chaperones in the ER or direct involvement of TRICB in procollagen folding. Our experiments indicated that the pathogenic effects of mutations in the transmembrane protein TAPT1 and in site-2 metalloprotease (S2P) might not be directly related to disruptions in synthesis, folding, or trafficking of procollagen chains. As expected, we found that patient cells with mutant P4H1 secreted abnormal procollagen that had significantly reduced thermal stability owing to under-hydroxylation of proline residues by P4H1. Surprisingly, however, we found no abnormalities in the procollagen folding or secretion rates, no evidence of misfolded procollagen accumulation in the cell, and no evidence of altered ER chaperone composition.

We also continued translational studies of OI caused by missense mutations in type I collagen that are not substitutions of obligatory Gly residues, specifically focusing on substitutions of Y-position arginine (Y-Arg) residues in the Gly-X-Y triplets within the collagen triple helix. We found that Y-Arg substitutions cause procollagen misfolding and accumulation in the ER to almost the same extent as Gly substitutions, because Y-Arg enhances collagen triple-helix stability and promotes triple-helix folding through binding of HSP47.

Presently, we are examining the molecular mechanism underlying OI caused by mutations in the ER–membrane stress receptor CREB3L1/OASIS. Preliminary analysis of RNA-seq and qPCR data combined with our new mRNA–based histopathology assay suggests common regulation CREB3L1 and some COPII proteins involved in secretory and/or autophagic procollagen export from the ER. However, the study is still in an early phase, and much work remains to be done.

Extracellular matrix pathology in tumors and fibrosis

Another aspect of our collagen metabolism pathology studies has been characterization of the pathology in fibromas and tumors, e.g., abnormal collagen composition of uterine fibroids and the potential role of type I collagen homotrimers in cancer. The normal isoform of type I collagen is a heterotrimer of two alpha1(I) chains and one alpha2(I) chain. Homotrimers of three alpha1(I) chains are produced in some fetal tissues, carcinomas, fibrotic tissues, as well as in rare forms of OI and EDS associated with alpha2(I) chain deficiency. We found the homotrimers to be at least 5–10 times more resistant to cleavage by all mammalian collagenases than the heterotrimers, and we determined the molecular mechanism of this resistance. Our studies suggested that cancer cells might utilize the collagen isoform to build collagenase-resistant tracks, thus supporting invasion through stroma of lower resistance.

We also investigated bone pathology and tumors caused by defects in cAMP signaling, e.g., associated with mutation in protein kinase A (PKA), which is a key enzyme in the cAMP signaling pathway. Initially, we studied synthesis of type I collagen homotrimers. However, over the last 3–5 years, our focus has shifted to abnormal differentiation of osteoblastic cells and deposition of bone. We found that knockouts of various PKA subunits cause not only abnormal organization and mineralization of bone matrix but also novel bone structures that had not been previously reported. For instance, we observed free-standing cylindrical bone spicules with an osteon-like organization of lamellae and osteocytes but an inverted mineralization pattern, a highly mineralized central core, and diminishing mineralization away from the central core. We assisted clinical researchers in characterizing abnormal osteoblast maturation, the role of an abnormal inflammatory response, and effects of anti-inflammatory drug treatments in such animals. Improved understanding of bone tumors caused by PKA deficiencies may not only clarify the role of cAMP signaling but may also suggest new approaches to therapeutic manipulation of bone formation in skeletal dysplasias.

Multimodal micro-spectroscopic imaging and mapping of tissues

Given that tissue analysis plays a crucial role in understanding and treating collagen metabolism disorders, we are developing methods to characterize not only cell function in tissue sections but also ECM composition and structure. Label-free micro-spectroscopic infrared and Raman imaging of tissues and cell cultures provides important information about the chemical composition, organization, and biological reactions inaccessible by traditional histology. By resolving the problem of light-path variations with passive thermomechanical stabilization, we developed high-definition (HD) infrared imaging and Raman micro-spectroscopic methods, achieving spectral reproducibility of up to two orders of magnitude better than with leading commercial instruments. The HD technology was essential for the analysis of abnormal collagen matrix deposition by CRTAP– and FKBP65–deficient cells. It has enabled us to assist NIBIB scientists in characterizing a functionalized carbon-nanotube approach to the delivery of anticancer agents into cells that overexpress hyaluronate receptors and is crucial to our current studies of bone structure and mineralization in the mouse models of OI and PKA deficiencies described above.

The power of the technology is illustrated by our studies of ECM structure and composition in a mouse model of diastrophic dysplasia (DTD). DTD is an autosomal recessive dysplasia that affects cartilage and bone development and is caused by mutations in the SLC26A2 sulfate transporter gene, deficient sulfate uptake by chondrocytes, and resulting under-sulfation of glycosaminoglycans in cartilage matrix. For instance, we found that chondroitin under-sulfation leads to disorientation of collagen fibers, disrupting a thin protective layer at the articular surface and causing subsequent cartilage degradation. We also investigated the relationship between chondroitin under-sulfation and the rate of its synthesis across the growing epiphyseal cartilage and built a mathematical model for the sulfation pathway, predicting treatment targets for sulfation-related chondrodysplasias and genes that might contribute to the juvenile idiopathic arthritis recently associated with single-nucleotide polymorphisms in SLC26A2. We are further extending the technology by combining imaging of bone and cartilage ECM composition and structure with biomechanical measurements at the same length scales and in vivo ECM studies at large scales by solid-state magnetic resonance imaging (MRI) being developed by our collaborators.

As a test of the technology and its important translational application, we are presently working on combining our advances in mRNA–based and micro-spectroscopy-based histopathological analysis for understanding normal growth plate homeostasis and growth plate pathology in OI. General growth deficiency and disproportional development of proximal and distal limb bones (rhizomelia) are common, clinically important, and yet poorly understood features of the disease. Spatially resolved imaging of mRNA at a single-cell level is enabling us to identify the progression of growth-plate chondrocytes through differentiation steps and expression of different ECM components at these steps. Spatially resolved micro-spectroscopic analysis of ECM organization at the same distance scales in the same tissue sections is enabling us to relate the cell differentiation and function to ECM composition, structure, and function.


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  2. Barnes AM, Ashok A, Makareeva EN, Bruseld M, Cabral WA, Weis MA, Moali C, Bettler E, Eyre DR, Cassella JP, Leikin S, Hulmes DJS, Kessler E, Marini JC. COL1A1 C-propeptide mutations cause ER mislocalization of procollagen and impair C-terminal procollagen processing. Biochim Biophys Acta Mol Basis Dis 2019;1865:2210–2223.
  3. Cabral WA, Fratzl-Zelman N, Weis M, Perosky JE, Alimasa A, Harris R, Kang H, Makareeva E, Barnes AM, Roschger P, Leikin S, Klaushofer K, Forlino A, Backlund PS, Eyre DR, Kozloff KM, Marini JC. Substitution of murine type I collagen A1 3-hydroxylation site alters matrix structure but does not recapitulate osteogenesis imperfecta bone dysplasia. Matrix Biol 2020;20-39.
  4. Omari S, Makareeva E, Gorrell L, Jarnik M, Lippincott-Schwartz J, Leikin S. Mechanisms of procollagen and HSP47 sorting during ER-to-Golgi trafficking. Matrix Biol 2020;93:79-94.
  5. Robey PG, Boskey AL, Leikin SL. The regulatory role of matrix proteins in mineralization of bone. Marcus and Feldman's Osteoporosis, 5th Edition 2020;Chapter 8:165-187.


  • Peter Basser, PhD, Section on Quantitative Imaging and Tissue Sciences, NICHD, Bethesda, MD
  • Carsten Bönnemann, MD, Neuromuscular and Neurogenetic Disorders of Childhood Section, NINDS, Bethesda, MD
  • Peter H. Byers, MD, University of Washington, Seattle, WA
  • Antonella Forlino, PhD, Università degli Studi di Pavia, Pavia, Italy
  • Ken Kozloff, PhD, University of Michigan, Ann Arbor, MI
  • Jennifer A. Lippincott-Schwartz, PhD, Howard Hughes Medical Institute, Janelia Research Campus, Ashburn, VA, and Adjunct Investigator, NICHD, Bethesda, MD
  • Fransiska Malfait, MD, PhD, Center for Medical Genetics, Ghent, Belgium
  • Joan C. Marini, MD, PhD, Section on Heritable Disorders of Bone and Extracellular Matrix, NICHD, Bethesda, MD
  • Pamela G. Robey, PhD, Craniofacial and Skeletal Diseases Branch, NIDCR, Bethesda, MD
  • Antonio Rossi, PhD, Università degli Studi di Pavia, Pavia, Italy
  • Constantine A. Stratakis, MD, D(med)Sci, Section on Endocrinology and Genetics, NICHD, Bethesda, MD
  • Sofie Symoens, PhD, Center for Medical Genetics, Ghent, Belgium


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