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National Institutes of Health

Eunice Kennedy Shriver National Institute of Child Health and Human Development

2019 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
  • Anna Roberts-Pilgrim, PhD, Postdoctoral Fellow
  • Laura Gorrell, BS, Predoctoral Fellow
  • Amanda L. Scheiber, MS, Special Volunteer
  • 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 forming 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. It 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 bone disorders

Osteoblasts and fibroblasts produce and secrete the massive amounts of type I procollagen needed to build the skeleton. 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 the conformation of natively folded human procollagen is less favorable than the unfolded one above 35°C. 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 responsible for degradation of misfolded molecules and forcing the cell to always function in a high-stress mode. Our findings indicate that one of the key factors in bone pathology is osteoblast malfunction resulting from excessive cell stress, which is often caused by increased procollagen misfolding, inability of the cell to handle the normal load of misfolded procollagen, or both.

The most common hereditary cause of elevated 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. Our studies of 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. For example, the first 85–90 amino acids at the N-terminal end of the triple helix form an "N-anchor" domain, mutations within which prevent normal N-propeptide cleavage. Incorporation of molecules with uncleaved N-propeptides into collagen fibrils leads to the hyperextensibility and joint laxity more characteristic of EDS.

Bone pathology associated with excessive procollagen misfolding of nonhereditary origin is likely to be more prevalent than OI. Indeed, our data suggest that such misfolding should occur upon changes in the osteoblast ER environment associated with aging, environmental factors, inflammation, etc. It is likely to contribute to age-related osteoporosis, bone loss during cancer treatment, and many other common ailments. However, almost nothing is known about the pathophysiology mechanism because procollagen folding and the consequences of its misfolding for the cell remain poorly understood.

Cell biology of procollagen misfolding

Our current research focuses on the cell biology of procollagen misfolding. In one approach, we are using 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) marked by the coat protein complex II (COPII). Contrary to widely held beliefs, however, these 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. Transport vesicle formation involves fusion of ER-Golgi Intermediate Compartment (ERGIC) membranes with ERES, which appears to facilitate HSP47 removal from procollagen. Misfolded procollagen is retained at ERES, resulting in a COPII–dependent modification of ERES membranes by ubiquitin and autophagic machinery. The resulting autophagic ERES are then directly engulfed by lysosomes and degraded.

This novel ERES microautophagy pathway for ERES–loaded cargo may have wide implications, given that COPII coat involvement in regulating autophagic degradation and cargo rerouting from the secretory to degradative pathway at ERES is likely to be a general rather than collagen-specific phenomenon. The hypothesis is currently under investigation in our and several collaborating laboratories. From clinical and translational perspectives, our findings may explain at least some of the pathologies in patients with COPII mutations as deficient autophagic degradation of difficult-to-fold proteins, another line of investigation in our and collaborating laboratories.

To validate the physiological significance and further build on these findings, we are expanding our tools 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 (site-directed recombination technology) target sites for manipulating the tag, e.g., changing the fluorescence color or completely replacing it. We demonstrated that the cells produce and mineralize bone-like ECM, enabling us to perform live-cell imaging of endogenous rather than transiently transfected procollagen. Presently, we are introducing additional Flp-recombinase target sites into the same gene to produce cell-culture models with a variety of different OI mutations. The same strategy can then be used to generate mouse models and to study other proteins.

In another approach, we are investigating the pathophysiology of the 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 from an Old Order Amish community in Pennsylvania. 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 investigating the mechanism and role of the cell stress in OI by altering how the cells adapt to it. Building on our success in understanding rerouting of misfolded procollagen from ERES to autophagic degradation, we examined how reduced autophagy, and therefore increased accumulation of misfolded procollagen in the ER, affected the severity of OI in G610C mice. Reduced expression of Atg5, a protein we found to be involved in enhancing ERES microautophagy, resulted in about 40% perinatal lethality of the animals, apparently owing to malfunction of lung fibroblasts. Given that 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. We also observed that cell-specific knock-out of the autophagy-related gene Atg5 in mature osteoblasts reduced bone synthesis and raised bone fragility, explaining the pathogenicity of misfolded procollagen accumulation in osteoblast ER in vivo.

New approaches to analysis and treatment of bone 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 treatment of cell malfunction caused by procollagen misfolding in cases that do not involve pathogenic mutations.

To pursue this 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. Our study on the effects of altered Atg5 expression on bone pathology in G610C mice confirmed that decreased autophagy worsens, and increased autophagy alleviates OI symptoms. However, we also found that osteoblasts degrade misfolded procollagen primarily by ERES microautophagy, which is only moderately enhanced rather than regulated by Atg5, 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 for characterizing 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 year, 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 coming to our laboratory to learn this approach, we hope that it will soon be adapted not only in research but also in 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), and 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 posttranslational 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 underhydroxylation 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 have found that Y-Arg substitutions cause procollagen misfolding and accumulation in the ER almost to 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 of 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, this 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 this 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 antiinflammatory drug treatments in these 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 microspectroscopic imaging and mapping of tissues

Given that tissue analysis plays 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 microspectroscopic 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 microspectroscopic 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 for 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 undersulfation of glycosaminoglycans in cartilage matrix. For instance, we found that chondroitin undersulfation 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 undersulfation 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 the gene encoding the SLC26A2 transporter. 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 important translational application, we are presently working on combining our advances in mRNA–based and microspectroscopy-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 this 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 microspectroscopic 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.


  1. Omari S, Makareeva E, Roberts-Pilgrim A, Mirigian L, Jarnik M, Ott C, Lippincott-Schwartz J, Leikin S. Noncanonical autophagy at ER exit sites regulates procollagen turnover. Proc Natl Acad Sci USA 2018;115:E10099-E10108.
  2. Makareeva E, Sun G, Mirigian LS, Mertz EL, Vera JC, Espinoza NA, Yang K, Chen D, Klein TE, Byers PH, Leikin S. Substitutions for arginine at position 780 in triple helical domain of the alpha1(I) chain alter folding of the type I procollagen molecule and cause osteogenesis imperfecta. PLoS One 2018;13:e0200264.
  3. Scheiber AL, Guess AJ, Kaito T, Abzug JM, Enomoto-Iwamoto M, Leikin S, Iwamoto M, Otsuru S. Endoplasmic reticulum stress is induced in growth plate hypertrophic chondrocytes in G610C mouse model of osteogenesis imperfecta. Biochem Biophys Res Commun 2019;509:235-240.
  4. Jayes FL, Liu B, Feng L, Aviles-Espinoza N, Leikin S, Leppert PC. Evidence of biomechanical and collagen heterogeneity in uterine fibroids. PLoS One 2019;14:e0215646.
  5. 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.


  • 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
  • Emilios K. Dimitriadis, PhD, Biomedical Engineering & Physical Science Shared Resource Program, NIBIB, Bethesda, MD
  • Antonella Forlino, PhD, Università degli Studi di Pavia, Pavia, Italy
  • Ferenc Horkay, PhD, Section on Quantitative Imaging and Tissue Sciences, NICHD, Bethesda, MD
  • 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
  • Charlotte L. Phillips, PhD, University of Missouri, Columbia, MO
  • 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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