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National Institutes of Health

Eunice Kennedy Shriver National Institute of Child Health and Human Development

2018 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

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 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. Given that 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 thus focuses primarily on the cell biology of procollagen misfolding. In one approach, we are collaborating with Jennifer Lippincott-Schwartz on using live-cell imaging to investigate the synthesis, folding, trafficking, and degradation of fluorescently tagged procollagen in osteoblasts. Live-cell imaging of osteoblasts transiently transfected with fluorescent procollagen chains revealed entirely unexpected features of procollagen quality control and trafficking. We observed sorting of normally folded and misfolded procollagen molecules at ER exit sites (ERES) marked by coat protein complex II (COPII). Normally folded procollagen was loaded into giant (up to 500 nm) Golgi-bound transport vesicles. Contrary to widely held beliefs, the vesicles do not have a COPII coat nor do they not contain HSP47, a collagen-specific ER chaperone that preferentially binds to natively folded procollagen to assist in its folding and loading into ERES. Apparently HSP47 is removed from procollagen upon its entry into ERES. Misfolded procollagen is retained at ERES, resulting in a COPII–dependent modification of ERES membrane by ubiquitin and autophagic machinery. The resulting autophagic ERES are subsequently directly engulfed by lysosomes and degraded. The findings delineate a novel, COPII–dependent, non-conventional micro-autophagy-like pathway for recycling ERES–loaded cargo [Reference 1].

Importantly, our findings may have wide implications beyond procollagen and ECM biology. For instance, COPII coat involvement in regulating autophagic degradation and cargo rerouting from the secretory to degradative pathway at ERES are likely to be general rather than collagen-specific phenomena. 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 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 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 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 to 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 micro-autophagy, 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.

Development of novel OI treatments

Our observations suggested that pathology associated with procollagen misfolding may be at least 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 potentially detrimental effects of secreted mutant collagen in OI, pharmacological treatment of cell malfunction is a more realistic short-term approach to OI than suppression of dominant negative OI mutations by gene therapy or bone marrow transplantation. Moreover, the same approach is likely to be a better long-term strategy for treatment of cell malfunction caused by procollagen misfolding in cases that do not involve pathogenic mutations.

In one approach to pursuing this strategy, we are targeting procollagen autophagy, given that enhancing the natural ability of the cell to remove and degrade the misfolded molecules via autophagy is the simplest way to prevent their pathogenic accumulation molecules 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. However, prolonged LPD stunted animal growth. We are thus developing intermittent LPD and fasting approaches that might provide the same benefits of autophagy enhancement without the detrimental side effects of long-term nutrient deprivation. At the same time, we are validating the general approach of autophagy enhancement by direct genetic modulation of autophagy efficiency. We were able to completely rescue the perinatal lethality in animals with altered endogenous Atg5 by introducing an Atg5 transgene that normalized the Atg5 protein level. The experiments suggested that the lethality was caused by quantitative Atg5 deficiency rather than other effects of the altered gene sequence and that increasing Atg5 expression can stimulate autophagy. We are completing the analysis of animals with transgenic Atg5 overexpression. So far, our observations are providing encouraging evidence of improvements in bone geometry in these animals. However, additional measurements are still required for fully understanding the Atg5 overexpression effects.

In another approach, we are collaborating with several extramural laboratories to test the effects of chemical chaperones, which are drugs known to reduce protein misfolding or the accumulation of misfolded proteins in the ER. In collaboration with Antonella Forlino, we found that 4-phenylbutyrate (4PBA) reduced OI severity in a zebrafish model. 4PBA is an FDA–approved scavenger of ammonia, but it also has chaperone and histone deacetylase inhibitor activities. It might also enhance autophagic degradation of misfolded procollagen, although the mechanism of this effect is presently unclear. In collaboration with Satoru Otsuru, we are testing effects of 4PBA on bone pathology in G610C mice. The study is still in its early stages, but preliminary data already indicate improved bone formation at least in 7-weak-old female animals.

Translational studies on patients with novel or unusual OI and EDS mutations

Abnormal collagen biosynthesis and malfunction of osteoblasts are also important factors in OI that is caused by other collagen mutations as well as by mutations in other proteins. Over the past several years, we assisted several clinical research groups in characterizing collagen biosynthesis and folding in fibroblasts from patients with newly discovered recessive forms of OI, with closely related skeletal dysplasias, and with more complex disorders that were 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). In particular, our collaboration with Joan Marini 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 role in procollagen folding remains unclear. More surprisingly, we found no detectable changes in the procollagen folding rate in cultured fibroblasts from patients with FKBP65 mutations. Our data suggest 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 syndrome), 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 [Reference 2]. 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 triple helix. In an earlier collaboration with Joan Marini, we found that Y-Arg substitutions with cysteine (Cys) caused procollagen misfolding, but it remained unclear whether the primary cause of misfolding was the loss of Y-Arg or aberrant disulfide bond formation by Cys. In a more recent collaboration with Peter Byers, we were able to examine cells from two different OI patients, one of whom had a Y-Arg substitution with Cys and the other a substitution of the same Y-Arg with leucine (Leu). The latter study revealed that the loss of Y-Arg, which enhances the collagen triple helix stability and promotes triple helix folding through binding of HSP47, disrupted procollagen folding and caused its accumulation in the ER to almost the same level as Gly substitutions [Reference 3].

Extracellular matrix pathology in tumors and fibrosis

Another important advance from our work of the past several years was the characterization of a collagenase-resistant, homotrimeric isoform of type I collagen and its potential role in cancer, fibrosis, and other disorders. 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 collaborated with Constantine Stratakis's lab to investigate bone tumors caused by defects in protein kinase A (PKA), a key enzyme in the cAMP signaling pathway. Initially, we investigated synthesis of type I collagen homotrimers. However, over the last 3–5 years, the focus of the study has shifted to abnormal differentiation of osteoblastic cells and deposition of bone within these tumors. 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 decreasing mineralization away from the central core. Currently, we are assisting the Stratakis lab in characterizing abnormal osteoblast maturation, the role of an abnormal inflammatory response, and effects of anti-inflammatory drug treatments in these animals [Reference 4]. 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.

Multi-modal micro-spectroscopic imaging and mapping of tissues

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. However, applications of these techniques were severely restricted by light-path instabilities in thin hydrated specimens under physiological conditions. We resolved the problem by designing specimen chambers with precise thermo-mechanical stabilization for high-definition (HD) infrared imaging and Raman micro-spectroscopy, achieving spectral reproducibility 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 best illustrated by our studies of ECM structure and of composition effects on the function of cartilage in a mouse model of diastrophic dysplasia (DTD), 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. In collaboration with Antonella Forlino and Antonio Rossi, we found that the deficiency results in under-sulfation of chondroitin and disorientation of collagen fibers, disrupting a thin protective layer at the articular surface and causing subsequent cartilage degradation. We investigated the relationship between chondroitin under-sulfation and the rate of its synthesis across the growing epiphyseal cartilage, and we 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 extending the technology by combining imaging of bone and cartilage ECM composition and structure with biomechanical measurements at the same length scales. The mechanical properties of bone and cartilage should depend on the deformation length scale because of the heterogeneous microscopic structure and the presence of various macroscopic regions and zones in these tissues. Nevertheless, biomechanical studies are rarely accompanied by mapping of tissue composition and structure. To address the problem, we are collaborating with Emilios Dimitriadis and Ferenc Horkay to map cartilage elasticity by force microscopy at length scales appropriate for examining the material properties of the ECM and to combine it with our multimodal imaging technology [Reference 5].

While the ECM plays a key role in normal development and pathology of all tissues, most studies focus on expression of its components rather than its overall organization. Our multimodal imaging technology is helping to close this gap in in vitro studies of tissue sections and cell cultures. To translate these advances into clinical practice, we are collaborating with Peter Basser on using the technology to calibrate and test newer methods for non-invasive in vivo ECM studies by the solid-state magnetic resonance imaging (MRI) that is being developed in his laboratory.


  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. Zou Y, Donkervoort S, Salo AM, Foley AR, Barnes AM, Hu Y, Makareeva E, Leach ME, Mohassel P, Dastgir J, Deardorff MA, Cohn RD, DiNonno WO, Malfait F, Lek M, Leikin S, Marini JC, Myllyharju J, Bonnemann CG. P4HA1 mutations cause a unique congenital disorder of connective tissue involving tendon, bone, muscle and the eye. Hum Mol Genet 2017;26:2207-2217.
  3. 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.
  4. Saloustros E, Liu S, Mertz EL, Bhattacharyya N, Starost MF, Salpea P, Nesterova M, Collins M, Leikin S, Stratakis CA. Celecoxib treatment of fibrous dysplasia (FD) in a human FD cell line and FD-like lesions in mice with protein kinase A (PKA) defects. Mol Cell Endocrinol 2017;439:165-174.
  5. Chandran PL, Dimitriadis EK, Mertz EL, Horkay F. Microscale mapping of extracellular matrix elasticity of mouse joint cartilage: an approach to extracting bulk elasticity of soft matter with surface roughness. Soft Matter 2018;14:2879-2892.


  • Peter Basser, PhD, Section on Quantitative Imaging and Tissue Sciences, NICHD, Bethesda, MD
  • Carsten Bonnemann, 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
  • Joan C. Marini, MD, PhD, Section on Heritable Disorders of Bone and Extracellular Matrix, NICHD, Bethesda, MD
  • Satoru Otsuru, MD, PhD, University of Maryland School of Medicine, Baltimore, 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


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