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Extracellular Matrix Disorders: Molecular Mechanisms and Treatment Targets

Sergey Leikin, PhD
  • Sergey Leikin, PhD, Head, Section on Physical Biochemistry
  • Edward L. Mertz, PhD, Staff Scientist
  • Elena N. Makareeva, PhD, Biologist
  • Lynn S. Mirigian (Felts), BS, Predoctoral Fellow (Graduate Student)

The extracellular matrix (ECM) 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. Our interest in ECM biology began with studies on basic principles relating the helical structure of collagen and DNA to their interactions and biological function. Over the years, the focus of our research shifted to collagens, which are the most abundant ECM molecules, and then to ECM disorders and the development of novel treatments for these disorders. We are gradually phasing out the studies on DNA interactions and concentrating on ECM pathology in cancer, fibrosis, osteogenesis imperfecta, Ehlers-Danlos syndrome, chondrodysplasias, osteoporosis, and other diseases. Together with other NICHD and extramural clinical scientists, we strive to improve our knowledge of the molecular mechanisms underlying these diseases. We hope to use the knowledge gained through these studies for diagnostics, characterization, and treatment, bringing our expertise in physical biochemistry and theory to clinical research and practice.

Procollagen folding and its role in bone disorders

Collagens are triple-helical proteins forming structural scaffolds that hold together bone, cartilage, skin, and other tissues. In bone, the proteins are produced by osteoblasts, the cells responsible for synthesis and mineralization of new bone material. By far the most abundant proteins in all vertebrates, they account for up to 30% of all proteins synthesized by osteoblasts. Yet, folding of their procollagen precursors within the endoplasmic reticulum (ER) presents an extraordinary challenge for cells. In particular, we discovered that the equilibrium state of type I procollagen in the absence of chaperones is a random coil. Folding of the human procollagen triple helix in an ER–like environment is favorable below 35°C but not at normal body temperature. At this temperature, natively folded collagen molecules become stable only after incorporation into fibers. When not incorporated into fibers, individual molecules denature within several hours and become susceptible to rapid proteolytic degradation. To overcome such intrinsic instability, cells must use specialized chaperones to fold the triple helix. It is not completely clear which chaperones are involved in this process, how they function, and how cells recognize properly folded procollagen and handle it when misfolded. Because of massive procollagen synthesis, osteoblast malfunction caused by procollagen misfolding might be an important factor in bone pathologies. For instance, osteogenesis imperfecta (OI) appears to be primarily a procollagen misfolding disease. Moreover, inability of aging osteoblasts to handle normal procollagen folding load might also contribute to common osteoporosis. Understanding procollagen folding and the cellular response to its misfolding is therefore important both from fundamental and practical perspectives.

The most common mutations affecting procollagen folding are Gly substitutions in the obligatory (Gly-X-Y) n sequence of the triple helix. For example, such substitutions in type I collagen are responsible for approximately 80% of severe OI cases. The substitutions' effects on procollagen folding are determined by the location and identity of the substituting residue. Our studies of collagens from OI patients with over 50 different Gly substitutions revealed several structural regions within the triple helix where mutations might be responsible for distinct OI phenotypes. In particular, the first 85–90 amino acids at the N-terminal end of the triple helix form an “N-anchor” domain with higher-than-average triple helix stability. Gly substitutions within this region disrupt the whole N-anchor, preventing normal cleavage of the adjacent N-propeptide. Incorporation of collagen with uncleaved N-propeptides into fibrils leads to hyperextensibility and joint laxity more characteristic of the Ehlers-Danlos syndrome (EDS). Gly substitutions within an α1(I) chain region surrounding the collagenase cleavage site might be lethal because the sequence of this region prevents efficient renucleation of the normal C-to-N-terminus helix folding, once the mutation is encountered.

At present, we are focusing on understanding how cells respond to misfolding of procollagen with different Gly substitutions. We hypothesize that unconventional procollagen triple helix folding requirements lead to unconventional ER stress response and to misfolding. In collaboration with Peter Byers, we found that Gly substitutions cause slower folding as well as misfolding of procollagen, its accumulation in the ER of cultured patient fibroblasts, ER stress, and upregulation of ER stress–regulated pro-apoptotic transcription factor CHOP (C/EBP homologous protein). Consistent with previous observations reported by us and others, we found no activation of the conventional unfolded protein response (UPR). Instead, quantitative real-time PCR (qPCR) evidence of NF-κB signaling activation pointed to possible similarity with ER–overload response to misfolding and polymerization of serpin-family proteins. NF-κB signaling activation is detrimental to osteoblast differentiation and function, possibly providing both an important clue to OI pathophysiology and an important therapeutic target.

As patient osteoblasts or bone marrow stromal cells (BMSCs) were not available, we further pursued this finding in a mouse model with a Gly610→Cys substitution in the α2(I) chain (Amish mouse). The model mimics the mutation found in a large group of patients from an Old Order Amish community in Lancaster County, PA. Our study of cultured dermal fibroblasts and calvarial osteoblasts from the Amish mouse model revealed misfolding of mutant molecules without activation of the UPR, in a highly similar manner to fibroblasts from human patients discussed above. We found that the cells preferentially secreted molecules without the mutant chains while retaining and degrading about 30% of the molecules with the mutant chain, which were apparently identified by the ER quality control as misfolded. The latter appeared to be degraded by lysosomes via autophagy rather than by proteasomes via the ER–associated degradation (ERAD) pathway more common in UPR. Similar observations were reported for fibroblasts expressing retrovirally introduced α1(I) chains with a Gly substitution instead of endogenous α1(I). In culture, calvarial osteoblasts from the Amish mouse were able to adapt to the misfolding of mutant molecules and effectively eliminate them by enhancing the autophagy. However, this adaptation occurred at the cost of abnormal cell function, e.g., blunted response to stimulation of collagen production by TGF-β. The adaptation also altered osteogenic differentiation of bone marrow stromal cells (BMSCs), which formed the same number of alkaline phosphatase–positive colonies but a substantially smaller number of colonies capable of producing mineralized matrix nodules. We observed increased nuclear localization of p65 in calvarial osteoblast and BMSC cultures from mutant animals, providing further evidence for involvement of the NF-κB pathway in osteoblast pathology. Electron microscopy of periosteal and trabecular osteoblasts in lumbar vertebrae sections revealed dramatic distention of ER cisternae and disruption of mitochondria, suggesting severe ER stress and reduced ability of osteoblasts to adapt to this stress in vivo. The experiments supported our hypothesis that changes in differentiation and function of osteoblasts in response to misfolding of mutant procollagen molecules play an important role in OI bone pathology.

Development of novel OI treatments

As an initial approach to further testing of this hypothesis and developing a treatment directed at the osteoblast ER stress, we selected a dietary intervention. We hypothesized that the severity of ER stress and the cost of cellular adaptation to it might be reduced by enhancing autophagy with a low protein diet. A low protein diet treatment of animals from 8 to 17 weeks of age reduced the swelling of ER cisternae in lumbar vertebrae osteoblasts and partially normalized mineralization of newly deposited cortical bones. However, these positive effects were counterbalanced by reduced animal growth and reduced deposition of new bone. Overall, our preliminary data suggest that autophagy enhancement is a useful therapeutic strategy, but a continuous low protein diet is not likely to be a suitable approach to achieving the autophagy enhancement, particularly in pediatric OI patients.

Concurrently, our study of calvarial cells and BMSCs revealed normalization of osteoblast differentiation and NF-κB signaling activation. Surprisingly, BMSCs appeared to "remember" the animal diet for up to several weeks at identical cell culture conditions and up to four passages. Apparently, the diet produced some epigenetic changes, e.g., resulting in enhanced autophagy even after the cell removal from animals and prolonged culture. A better understanding of the changes and their effects on cellular differentiation and function might enable us to use an intermittent low protein diet as an alternative approach to alleviating the ER stress in osteoblasts without reducing bone deposition. At present, we are continuing to analyze the changes in cellular differentiation and function associated with the diet and are testing alternative, pharmacological approaches to autophagy enhancement and/or suppression of abnormal NF-κB signaling activation in osteoblasts. Our results are still preliminary and more experiments remain to be completed.

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

Abnormal collagen biosynthesis and malfunction of osteoblasts are also important factors in OI caused by other mutations in collagen as well as by mutations in other proteins. Over the last 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 caused by mutations in cartilage-associated protein (CRTAP), prolyl-3-hydrohylase (P3H1), cyclophilin B (CYPB), the molecular chaperone FKBP65, and WNT1 (1). In particular, 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 role in procollagen folding remains unclear.

More recently, in collaboration with Joan Marini, we investigated collagen and ECM biosynthesis abnormalities caused by CYPB and FKBP65 mutations. In patient fibroblasts that did not translate CYPB, we found the procollagen folding rate to be slower. More detailed follow-up studies of cells and tissues from CYPB–deficient mice created by Marini showed an altered pattern of lysine hydroxylation and glycosylation, decreased ECM deposition by cultured cells, and abnormal collagen crosslinking in the ECM. In cultured fibroblasts from patients with FKBP65 mutations, we found no detectable changes in procollagen folding rate (2). We observed significant abnormalities in deposition and crosslinking of ECM, which likely contribute to joint contractures in some of the patients (3). It remains unclear why some FKBP65 mutations cause severe OI with joint contractures (Bruck syndrome) while other mutations cause joint contractures without pronounced OI (Kuskokwim syndrome) (3) or OI without pronounced joint contractures.

A particularly puzzling form of OI is associated with deficiencies in the pigment epithelium–derived factor (PEDF). The protein was initially described as an anti-angiogenesis factor, but the loss of function of both PEDF alleles leads to severe, progressively deforming OI (type VI) rather than out-of-control angiogenesis. In collaboration with Frank Rauch, we investigated procollagen biosynthesis and ECM deposition by cultured fibroblasts from patients with type VI OI, but detected no clear abnormalities. In collaboration with Patricia Becerra, we found that PEDF is capable of completely inhibiting collagen fibrillogenesis at concentrations comparable to its physiological level in serum, but it is presently unclear whether this PEDF property is physiologically important.

Most single amino acid substitutions in X and Y positions of the repeating Gly-X-Y collagen sequence are considered to be neutral variants. Several Arg-to-Cys substitutions were recently reported, but it remained unclear whether the resulting OI and EDS were caused by aberrant disulfide bonds formed in the ECM by the Cys residue, which is not present in normal type I collagen, or by abnormal biosynthesis of mutant molecules (29-34). In collaboration with Peter Byers, we analyzed collagen metabolism pathology caused by substitutions of the same Y-position Arg780 in the α1(I) chain to Leu and Cys. The study revealed that the loss of Y-position Arg causes abnormal procollagen folding by reducing triple helix stability and eliminating important HSP47 binding sites. The effects are likely responsible for the bone fragility caused by Y- but not X-position Arg substitutions, while aberrant Cys disulfide bonds likely contribute to EDS in patients with either Y- or X- position substitutions.

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 α1(I) chains and one α2(I) chain. Homotrimers of three α1(I) chains are produced in some fetal tissues, carcinomas, fibrotic tissues, as well as in rare forms of OI and EDS associated with α2(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 this collagen isoform for building collagenase-resistant tracks, supporting invasion through stroma of lower resistance. Importantly, cancer cells produce cancer-specific isoforms of trypsin and other enzymes with altered post-translational modification. It was proposed that cancer-specific trypsins are capable of cleaving collagen triple helices at multiple sites. However, we found that such literature reports reflected artifacts of collagen degradation during denaturation for gel electrophoresis. Cancer-associated trypsins are not capable of cleaving the native collagen triple helix, yet they likely contribute to ECM degradation through solubilizing collagen molecules by cleaving the telopeptides involved in intermolecular cross-links (4).

We also collaborated with Program on Developmental Endocrinology and Genetics (PDGEN) scientists at NICHD to investigate bone tumors caused by defects in protein kinase A (PKA), a crucial enzyme in the cAMP signaling pathway. Initially, we investigated synthesis of type I collagen homotrimers. However, over the last two years, the focus of this 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 reported before. For instance, we observed free-standing cylindrical bone spicules with osteon-like organization of lamellae and osteocytes but inverted mineralization pattern, highly mineralized central core, and decreasing mineralization away from the central core. Currently, we are assisting PDGEN scientists in characterizing abnormal osteoblast maturation, the role of abnormal inflammatory response, and effects of anti-inflammatory drug treatments in these animals. Improved understanding of bone tumors caused by PKA deficiencies may not only clarify the role of cAMP signaling but 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 this problem by designing specimen chambers with precise thermo-mechanical stabilization for high-definition (HD) infrared imaging and Raman micro-spectroscopy, achieving the spectral reproducibility up to two orders of magnitude better than in leading commercial instruments. The HD technology was essential for analysis of abnormal collagen matrix deposition by CRTAP– and FKBP65–deficient cells (1–3). It enabled us to assist NIBIB scientists in characterizing a functionalized carbon nanotube approach to delivery of anticancer agents into cells that overexpress hylauronate receptors. It is also crucial for our current studies of bone structure and mineralization in the mouse models of OI and PKA deficiencies described above.

The power of this technology is best illustrated by our studies of ECM structure and composition effects on the function of cartilage in a mouse model of diastrophic dysplasia (DTD) (5). DTD is a recessive chondrodysplasia caused by mutations in the SLC26A2 sulfate transporter, which lead to deficient sulfate uptake by chondrocytes and 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. By combining HD infrared and micro-radiographic 35S imaging, we observed a correlation between the extent of chondroitin under-sulfation and the rate of its synthesis across the growing epiphyseal cartilage. Based on this correlation and literature data, 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 SLC26A2 transporter.

We are extending this 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 different 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 Peter Basser and Emilios Dimitriadis on mapping of cartilage elasticity by force microscopy at length scales appropriate for examining the material properties of ECM and on combining it with our multimodal imaging technology.


  1. Valli M, Barnes AM, Gallanti A, Cabral WA, Viglio S, Weis MA, Makareeva E, Eyre D, Leikin S, Antoniazzi F, Marini JC, Mottes M. Deficiency of CRTAP in non-lethal recessive osteogenesis imperfecta reduces collagen deposition into matrix. Clin Genet 2012;82:453-459.
  2. Barnes AM, Cabral WA, Weis M, Makareeva E, Mertz EL, Leikin S, Eyre D, Trujillo C, Marini JC. Absence of FKBP10 in recessive type XI osteogenesis imperfecta leads to diminished collagen cross-linking and reduced collagen deposition in extracellular matrix. Hum Mutat 2012;33:1589-1598.
  3. Barnes AM, Duncan G, Weis M, Paton W, Cabral WA, Mertz EL, Makareeva E, Gambello MJ, Lacbawan FL, Leikin S, Fertala A, Eyre DR, Bale SJ, Marini JC. Kuskokwim Syndrome, a Recessive Congenital Contracture Disorder, Extends the Phenotype of FKBP10 Mutations. Hum Mutat 2013;34:1279-1288.
  4. Mirigian LS, Makareeva E, Koistinen H, Itkonen O, Sorsa T, Stenman UH, Salo T, Leikin S. Collagen degradation by tumor-associated trypsins. Arch Biochem Biophys 2013;535:111-114.
  5. Mertz EL, Facchini M, Pham AT, Gualeni B, De Leonardis F, Rossi A, Forlino A. Matrix disruptions, growth, and degradation of cartilage with impaired sulfation. J Biol Chem 2012;287:22030-22042.


  • Peter Basser, PhD, Program on Pediatric Imaging and Tissue Sciences, NICHD, Bethesda, MD
  • S. Patricia Becerra, PhD, Laboratory of Retinal Cell and Molecular Biology, NEI, Bethesda, MD
  • Peter H. Byers, MD, University of Washington, Seattle, WA
  • Sarah Dallas, PhD, University of Missouri, Kansas City, MO
  • Emilios K. Dimitriadis, PhD, Biomedical Engineering & Physical Science Shared Resource Program, NIBIB, Bethesda, MD
  • Motomi Enomoto-Iwamoto, DDS, PhD, >University of Pennsylvania, Philadelphia, PA
  • Antonella Forlino, PhD, Università degli Studi di Pavia, Pavia, Italy
  • Kenn Holmbeck, PhD, Matrix Metalloproteinase Section, NIDCR, Bethesda, MD
  • Alexei A. Kornyshev, PhD, Imperial College, University of London, London, UK
  • Ken Kozloff, PhD, University of Michigan, Ann Arbor, MI
  • Joan C. Marini, MD, PhD, Bone and Extracellular Matrix Branch, NICHD, Bethesda, MD
  • George Patterson, PhD, Section on Biophotonics, NIBIB, Bethesda, MD
  • Charlotte L. Phillips, PhD, University of Missouri, Columbia, MO
  • Frank Rauch, MD, Shriners Hospital for Children, Montreal, Canada
  • Pamela G. Robey, PhD, Craniofacial and Skeletal Diseases Branch, NIDCR, Bethesda, MD
  • Antonio Rossi, PhD, Università degli Studi di Pavia, Pavia, Italy
  • Dan L. Sackett, PhD, Program in Physical Biology, NICHD, Bethesda, MD
  • Constantine A. Stratakis, MD, DSci, Program in Developmental Endocrinology and Genetics, NICHD, Bethesda, MD


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