A. Sampath Narayanan

X-Linked Cutis Laxa: Defective Cross-Link Formation in Collagen Due to Decreased Lysyl Oxidase Activity

We studied several members of a family with an X-linked form of cutis laxa; the affected males have mild skin laxity, a characteristic facies, skeletal abnormalities, structural abnormalities of the genitourinary tract, and low serum copper levels. The activity of lysyl oxidase, a copper-dependent enzyme involved in cross-link formation in collagen, was decreased in skin-biopsy specimens (13 to 26 per cent of normal) and in culture medium from cells to two affected males (15 to 20 per cent of normal). Immunoreactive lysyl oxidase from skin of both patients was virtually undetectable by immunodiffusion assay. The amounts of lysyl-derived aldehydes (the product formed in collagen and elastin by lysyl oxidase) and of cross-links formed from these products were decreased in dermal fibroblasts in culture. Collagen extractability from these cells was increased in culture. These findings suggest that lysyl oxidase deficiency provides the biochemical basis of the X-linked form of cutis laxa.

Connective Tissues of the Periodontium: A Summary of Current Work

Abstract The connective tissues around the teeth have been studied extensively with regard to their construction, composition, and metabolism under both normal and pathologic conditions. These tissues have served as a model for connective tissues in general, especially in studies aimed at understanding mechanisms regulating normal tissue maintenance and turnover, and changes which occur as disease develops. In normal gingiva, the proportions of the various collagen types are very similar to those in skin, but the molecules appear to be tailored differently. After the teeth are removed, the gingiva becomes converted to mucosa, and the characteristics of the collagens change. As periodontitis develops, significant qualitative and quantitative changes occur. The rate of collagen production and degradation are greatly increased, while the total amount of collagen decreases. The amount of type V collagen increases about seven-fold, and an entirely new collagen, the type I trimer, appears which may account for up to 29% of the total collagen present. The types and proportions of collagens synthesized by fibroblasts from normal gingiva are very similar to those found in the normal tissue. Fibroblasts derived from inflamed gingiva are a unique phenotype in that they continue to synthesize the type I trimer in vitro, and it may account for up to 14% of the total collagen produced in some cultures. Cells obtained from explants of tissue from individuals with drug-induced or spontaneously occurring gingival hyperplasia are also unusual; they synthesize abnormal amounts of protein and collagen, and these characteristics persist indefinitely in vitro. Fibroblast functions are regulated by environmental ligands, some found in blood platelets and plasma, and others synthesized and secreted by infiltrating leukocytes. For example, substances present in serum cause the cells to decrease their production of type III collagen, while greatly increasing production of type V. The C1 component of complement is a mitogen for a subpopulation of gingival fibroblasts, and the cells display binding sites for the collagenous region of the C1q subcomponent of C1. Activated lymphoid cells release a factor or factors which inhibit cell growth by preventing cells in the G0 or G1 stage of the cell cycle from leaving, and by preventing division of cells in G2. In these cells, total protein synthesis is affected only slightly, but collagen synthesis is greatly inhibited; in addition, the production of an unidentified protein of Mr approximately 70,000-daltons appears to be halted. Activated macrophages also produce substances which affect fibroblasts, among which are the prostaglandins. PGE2 interacts with a subpopulation of gingival fibroblasts in culture to inhibit membrane transport, protein synthesis, and growth without apparent effects on the remaining cells. In contrast, epidermal growth factor enhances growth and total protein production, without affecting the amount of collagen synthesized. The mechanisms underlying the observed pathologic alterations are complex and not well understood. Differential degradation by collagenase and other enzymes present in inflamed tissues may account in part for the changes in the quantity and ratios of the collagens. The amounts and types of regulatory molecules in inflamed and non-inflamed tissue differ greatly, and this may alter fibroblast growth and synthesis activities. However, these processes cannot account for the presence of unusual phenotypes in diseased tissue, nor for their persistence indefinitely in culture. To account for these observations, we have proposed that human fibroblasts are genetically heterogeneous, that a process of clonal selection governed by environmental ligands which inhibit or enhance growth rates occurs, and that the tissue composition observed in both normal and diseased tissue is in part a consequence of the mixtures of clones present.

Progenitor cells from dental follicle are able to form cementum matrix in vivo

To address the molecular mechanisms of cementogenesis, we have isolated dental follicle cells and examined them to see if they contain cementoblast progenitors. Dental follicle tissue was dissected from the root surface of bovine tooth germ and cells were released by digestion with bacterial collagenase. The released cells were maintained as a bovine dental follicle cells (BDFC). To elucidate the differentiation capacity of BDFC, they were transplanted into severe combined immunodeficiency (SCID) mice for 4 weeks. Transplanted BDFC formed cementumlike matrix; in contrast, bovine alveolar osteoblast (BAOB) transplants formed bonelike matrix, and bovine periodontal ligament cells (BPDL) formed a small amount of the cementumlike matrix. Immunohistochemical analysis showed that cementumlike matrix was positive for anti-cementum attachment protein monoclonal antibody, whereas bonelike matrix was negative. These results indicated that the BDFC contained cementoblast progenitors that were able to differentiate to cementoblasts in vivo. They also indicated that the BDFC are phenotypically distinct from BAOB and BPDL, and provide a useful model for investigating molecular mechanisms of cementogenesis.

Cementum matrix formation in vivo by cultured dental follicle cells

Dental follicle is the fibrous tissue that surrounds the developing tooth germ, and it is believed to contain progenitors for cementoblasts, periodontal ligament cells, and osteoblasts. In this study, we report the presence of cementoblast progenitors in cultures of bovine dental follicle cells and demonstrate their differentiation capacity. Bovine dental follicle cells (BDFC) obtained from tooth germs by collagenase digestion were compared with bovine alveolar bone osteoblasts (BAOB) and bovine periodontal ligament cells (BPDL) in vitro and in vivo. In culture, BDFC exhibited low levels of alkaline phosphatase activity and expressed mRNA for osteopontin (OP) and type I collagen (COLI), as well as low levels of osteocalcin (OC) mRNA. In contrast, cultured BAOB exhibited high alkaline phosphatase activity levels and expressed mRNA for OC, OP, COLI, and bone sialoprotein (BSP). To elucidate the differentiation capacity of BDFC in vivo, cells were transplanted into severe combined immunodeficiency (SCID) mice and analyzed after 4 weeks. Transplanted BDFC formed fibrous tissue and cementum-like matrix, which stained positive for anti-cementum attachment protein (CAP) monoclonal antibody (3G9), and expressed mRNA for OC, OP, COLI, and BSP. On the other hand, transplanted BAOB formed bone-like matrix, but were negative for anti-CAP monoclonal antibody. The BPDL transplants formed fibrous tissue that contained a few cells expressing CAP. These results indicate that cementoblast progenitors are present in BDFC, which can provide a useful model for investigating the molecular mechanisms of cementogenesis.

A collagenous cementum-derived attachment protein is a marker for progenitors of the mineralized tissue-forming cell lineage of the periodontal ligament

The periodontal ligament (PDL) is a fibrous and cellular connective tissue that mediates tooth attachment to bone, and it comprises fibroblastic and mineralized tissue‐forming (MTF) progenitors. The MTF progenitors are believed to give rise to the cementoblastic and osteoblastic lineages. Cementum attachment protein (CAP) is a collagenous cementum‐derived protein which binds strongly to osteoblasts, moderately to PDL cells, and weakly to gingival fibroblasts. The aim of the present study was to determine the relationship between the capacity of PDL progenitors to bind CAP and their potential to express alkaline phosphatase (ALP) and form mineralized‐like tissue in culture. Cloned human PDL progenitor populations obtained from nine human donors were assayed for their constitutive capacity to bind CAP and express ALP, and for the dexamethasone‐induced potential to form mineralized‐like tissue in culture in the presence of ascorbic acid and β‐glycerophosphate. Forty percent of the progenitor clones produced mineralized‐like tissue. Two patterns of mineralization were observed: a spread and flat pattern similar to that produced by human bone cells in culture and a nodular ridge–like type resembling that formed by human cementoma‐derived cells. A direct correlation was found between the percentage of ALP positive cells in each progenitor clone and the amount of mineralized‐like tissue formed (r = 0.565). Similar correlations were found between the number of ALP positive cells and the binding capacity of each clone (r = 0.392) and between the CAP binding capacity and mineralized‐like tissue formation (r = 0.584). Multiple regression analysis indicated that the constitutive capacity of a clone to bind CAP and express ALP is directly correlated to its dexamethasone‐induced potential to form mineralized tissue (r = 0.675). These results indicate that CAP binding and ALP expression can serve as markers for the identification of MTF progenitors in the heterogeneous cultured population of the human periodontal ligament. These data show for the first time that binding capacity to extracellular components of mineralized tissues can be a marker for mineralized tissue‐forming progenitors.

Promotion of selective cell attachment by the RGD sequence in dentine matrix protein 1

Dentine matrix protein 1 (DMP1) is an important component of the non-collagenous extracellular matrix of developing teeth and bones. Functions of DMP1 other than a putative role in the initiation of mineralization are largely unknown. A first report on the DNA and deduced amino acid sequence showed that DMP1 has a single Arg-Gly-Asp (RGD) sequence. Here, whether the RGD sequence functions as a cell-attachment domain was tested. Using site-directed mutagenesis, two mutant recombinant DMP1 proteins with specific alterations at the RGD site were created. In the first mutant protein the RGD sequence was altered to a RGE (RGE) sequence; in the second the RGD domain was deleted (DEL). Mutated proteins were confirmed to be DMP1 by partial protein sequencing and dot-blot analysis with an anti-DMP1 antibody. Attachment of RPC-C2A (dental pulp cells), MC3T3-E1 (calvarial cells) or CHO (Chinese hamster ovary cells) to non-tissue-culture plastic coated with either DMP1, RGE or DEL proteins was compared. Bovine serum albumin and fibronectin served as negative and positive controls, respectively. The RGD-containing native DMP1 protein effectively allowed cell attachment and spreading. The RGE and DEL proteins with the altered and deleted RGD sites were significantly less effective in promoting cell attachment than the recombinant DMP1. Both RPC-C2A pulp cells and MC3T3-E1 cells showed similar reductions in attachment to mutated proteins. Treatment of RPC-C2A cells with a RGD-containing peptide prior to plating on DMP1-coated chambers abolished DMP1-mediated cell attachment. In contrast to RPC-C2A and MC3T3-E1cells, CHO cells, which normally do not express DMP1, failed to attach to DMP1. These data demonstrate that DMP1 promotes cell attachment through the RGD domain and that the attachment is cell- and tissue-specific. A basis for these observations is proposed using computer-generated models of the polypeptides within the DMP1 protein containing the RGD, RGE or DEL sequences.

Serum modulates collagen types in human gingiva fibroblasts.

Collagen is the most abundant structural protein of connective tissues. At least four different collagen types have been described so far. The tissue content and ratio of collagens are altered in pathologic states such as scleroderma and atherosclerosis, as well as during normal growth [l-3] . However, very little is known about the factors which influence the rates of synthesis and degradation of collagen or the molecular species produced. Serum factors have been known to regulate cell function by effecting various cellular processes such as cell movement, viability and proliferation [4], and recently they have been implicated in the pathogenesis of certain fibrotic lesions such as atherosclerosis [5]. We have studied the effect of serum on collagen synthesis by human gingiva fibroblasts and we report that serum enhances type I collagen several-fold; however, the type III collagen is increased only slightly, and as a result, its proportion decreases with increasing serum concentration.

Effect of epidermal growth factor on the synthetic activity of human fibroblasts

The influence of epidermal growth factor on DNA and protein synthesis by human gingival fibroblasts was studied. Synchronized cells treated with epidermal growth factor synthesized considerably greater amounts of DNA relative to 10% fetal calf serum and the peak of synthesis ocurred 6 h later than with serum. Epidermal growth factor caused a dose-dependent stimulation of protein synthesis (proline incorporation). Collagen synthesis remained unaffected and, as a result, the proportion of collagen synthesized decreased with increasing epidermal growth factor concentration. Aspirin and indomethacin did not abrogate these effects, indicating that prostaglandins may not be involved.

Stability and purification of lysyl oxidase.

Abstract Previous attempts to purify lysyl oxidase have been frustrated by the failure to recover activity during ion exchange or affinity chromatography. We have found that lysyl oxidase from chick cartilage shows marked stability in buffers containing urea and in these solutions can be recovered in high yield from DKAE-cellulose and collagen-derivatized Sepharose. The purified enzyme was active against both collagen and elastin substrates but devoid of monoamine oxidase activity. An absolute requirement for oxygen for activity was found.

Cementum protein 1 (CEMP1) induces a cementoblastic phenotype and reduces osteoblastic differentiation in periodontal ligament cells

Cementum is a calcified tissue covering the tooth root surface, which functions as rigid tooth‐anchoring structure. Periodontal ligament is a unique non‐mineralized connective tissue, and is a source of mineralized tissue forming cells such as cementoblasts and osteoblasts. The CEMP1 is a novel cementum component the presence of which appears to be limited to cementoblasts and their progenitors. In order to understand the function of CEMP1, we investigated CEMP1 expression during the differentiation of human periodontal ligament cells. Immunomagnetically enriched alkaline phosphatase (ALP)‐positive periodontal ligament cells preferentially expressed CEMP1. CEMP1 expression was reduced when periodontal ligament cells differentiated to osteoblasts in vitro. Over‐expression of CEMP1 in periodontal ligament cells enhanced cementoblast differentiation and attenuated periodontal and osteoblastic phenotypes. Our data demonstrate for the first time that the CEMP1 is not only a marker protein for cementoblast‐related cells, but it also regulates cementoblast commitment in periodontal ligament cells. J. Cell. Physiol. 227: 649–657, 2012.