Quantitative analysis of DHEA and androsterone in female urine: investigating the effects of menstrual cycle, oral contraception and training on exercise-induced changes in young women.

Dehydroepiandrosterone (DHEA) and its metabolite androsterone (A) are natural steroids secreted in high quantities in human body. To assess the influence of oral contraceptives, menstrual cycle phase, and also physical exercise (acute and chronic such as training) on these metabolites excretions, a collection of 28 female urine specimens was organized. A three-extraction-step method was developed, and the analyses were performed by gas chromatography-mass spectrometry using deuterated 19-noretiocholanolone as the internal standard. Sample hydration state was found to be of great importance for kinetic studies, as it directly influenced the concentrations. No influence of menstrual cycle and training was found for androsterone and DHEA. However, oral contraceptive intake lowered DHEA excretion in urine and A seems to be slightly affected by exercise.

Optimizing the extraction and analysis of DHEA sulfate, corticosteroids and androgens in urine: application to a study of the influence of corticosteroid intake on urinary steroid profiles.

A method of detecting and quantifying dehydroepiandrosterone (DHEA) sulfate, corticosteroids, and androgens has been developed. All of the compounds were first extracted from urine using solid phase extraction (SPE), enzymatically hydrolyzed, and separated into three samples using a second SPE. A DHEA sulfate sample was acetylated and re-extracted using SPE for purification before analysis. Corticosteroid samples were oxidized and re-extracted using liquid/liquid extraction for analysis. Androgen samples were acetylated and re-extracted using SPE prior to analysis. The extraction and analysis methods were investigated and optimized. Analyses were performed with gas chromatography/mass spectrometry (GC/MS) and gas chromatography/flame ionization detection (GC/FID). The entire procedure was then applied to the study of urine profiles of healthy volunteers and patients treated with corticosteroids. The results showed that the quantities of androgens found in patient urines were lower than in those of healthy volunteers. In addition, other metabolites were detected in patient urines.

Delayed wound repair and impaired angiogenesis in mice lacking syndecan-4.

The syndecans make up a family of transmembrane heparan sulfate proteoglycans that act as coreceptors with integrins and growth factor tyrosine kinase receptors. Syndecan-4 is upregulated in skin dermis after wounding, and, in cultured fibroblasts adherent to the ECM protein fibronectin, this proteoglycan signals cooperatively with beta1 integrins. In this study, we generated mice in which the syndecan-4 gene was disrupted by homologous recombination in embryonic stem cells to test the hypothesis that syndecan-4 contributes to wound repair. Mice heterozygous or homozygous for the disrupted syndecan-4 gene are viable, fertile, and macroscopically indistinguishable from wild-type littermates. Compared with wild-type littermates, mice heterozygous or homozygous for the disrupted gene have statistically significant delayed healing of skin wounds and impaired angiogenesis in the granulation tissue. These results indicate that syndecan-4 is an important cell-surface receptor in wound healing and angiogenesis and that syndecan-4 is haplo-insufficient in these processes.

Growth and development of the human cricoid cartilage: an immunohistochemical analysis of the maturation sequence of the chondrocytes and surrounding cartilage matrix.

OBJECTIVES: The goal was to determine maturational changes in the human cricoid cartilage. STUDY DESIGN: The study involved immunohistochemical staining of collagen II (a marker of proliferating chondrocytes), matrilin-1 (a marker of post-proliferative chondrocytes), and collagen X (a marker of hypertrophic chondrocytes). Specimens included uninjured human cricoid cartilages at 18 and 41 weeks' gestation and 1, 4, and 13 years postpartum. RESULTS: This study demonstrated that type II collagen peaks in concentration at approximately 41 weeks' gestation. Matrilin-1 is present in progressively lower concentration in the central core of the cricoid ring, but the peripheries of the ring contain the protein in relatively high concentration. Type X collagen is not expressed in the age groups tested. CONCLUSIONS: These biochemical markers lend further support to a chondrocyte proliferative phase that slows between 1 and 4 years of age. Chondrocytes then enter a phase histologically similar to the hypertrophic phase but are biochemically different than hypertrophic chondrocytes destined for endochondral ossification.

Syndesmos, a protein that interacts with the cytoplasmic domain of syndecan-4, mediates cell spreading and actin cytoskeletal organization.

Syndecan-4 is a cell surface heparan sulfate proteoglycan which, in cooperation with integrins, transduces signals for the assembly of focal adhesions and actin stress fibers in cells plated on fibronectin. The regulation of these cellular events is proposed to occur, in part, through the interaction of the cytoplasmic domains of these transmembrane receptors with intracellular proteins. To identify potential intracellular proteins that interact with the cytoplasmic domain of syndecan-4, we carried out a yeast two-hybrid screen in which the cytoplasmic domain of syndecan-4 was used as bait. As a result of this screen, we have identified a novel cellular protein that interacts with the cytoplasmic domain of syndecan-4 but not with those of the other three syndecan family members. The interaction involves both the membrane proximal and variable central regions of the cytoplasmic domain. We have named this cDNA and encoded protein syndesmos. Syndesmos is ubiquitously expressed and can be myristylated. Consistent with its myristylation and syndecan-4 association, syndesmos colocalizes with syndecan-4 in the ventral plasma membranes of cells plated on fibronectin. When overexpressed in NIH 3T3 cells, syndesmos enhances cell spreading, actin stress fiber and focal contact formation in a serum-independent manner.

Syndecan-4 core protein is sufficient for the assembly of focal adhesions and actin stress fibers.

The formation of focal adhesions and actin stress fibers on fibronectin is dependent on signaling through (&bgr;)1 integrins and the heparan sulfate proteoglycan syndecan-4, and we have analyzed the requirement of the glycosaminoglycan chains of syndecan-4 during these events. Chinese hamster ovary cells with mutations in key enzymes of the glycanation process do not synthesize glycosaminoglycan chains and are unable to assemble actin stress fibers and focal contacts when cultured on fibronectin. Transfection of the mutant cells with a cDNA that encodes the core protein of chicken syndecan-4 leads to the production of unglycanated core protein. The overexpression of syndecan-4 core protein in these mutant cells increases cell spreading and is sufficient for these cells to assemble actin stress fibers and focal adhesions similar to wild-type cells seeded on fibronectin and vitronectin matrices. Syndecan-4 core protein colocalizes to focal contacts in mutant cells that have been transfected with the syndecan-4 core protein cDNA. These data indicate an essential role for the core protein of syndecan-4 in the generation of signals leading to actin stress fiber and focal contact assembly.

Assembly of a novel cartilage matrix protein filamentous network: molecular basis of differential requirement of von Willebrand factor A domains.

Cartilage matrix protein (CMP) is the prototype of the newly discovered matrilin family, all of which contain von Willebrand factor A domains. Although the function of matrilins remain unclear, we have shown that, in primary chondrocyte cultures, CMP (matrilin-1) forms a filamentous network, which is made up of two types of filaments, a collagen-dependent one and a collagen-independent one. In this study, we demonstrate that the collagen-independent CMP filaments are enriched in pericellular compartments, extending directly from chondrocyte membranes. Their morphology can be distinguished from that of collagen filaments by immunogold electron microscopy, and mimicked by that of self-assembled purified CMP. The assembly of CMP filaments can occur from transfection of a wild-type CMP transgene alone in skin fibroblasts, which do not produce endogenous CMP. Conversely, assembly of endogenous CMP filaments by chondrocytes can be inhibited specifically by dominant negative CMP transgenes. The two A domains within CMP serve essential but different functions during network formation. Deletion of the A2 domain converts the trimeric CMP into a mixture of monomers, dimers, and trimers, whereas deletion of the A1 domain does not affect the trimeric configuration. This suggests that the A2 domain modulates multimerization of CMP. Absence of either A domain from CMP abolishes its ability to form collagen-independent filaments. In particular, Asp22 in A1 and Asp255 in A2 are essential; double point mutation of these residues disrupts CMP network formation. These residues are part of the metal ion-dependent adhesion sites, thus a metal ion-dependent adhesion site-mediated adhesion mechanism may be applicable to matrilin assembly. Taken together, our data suggest that CMP is a bridging molecule that connects matrix components in cartilage to form an integrated matrix network.

Syndecan-4 signals cooperatively with integrins in a Rho-dependent manner in the assembly of focal adhesions and actin stress fibers.

The assembly of focal adhesions and actin stress fibers by cells plated on fibronectin depends on adhesion-mediated signals involving both integrins and cell-surface heparan sulfate proteoglycans. These two cell-surface receptors interact with different domains of fibronectin. To attempt to identify the heparan sulfate proteoglycans involved, we used fibronectin-null (FN-/-) mouse fibroblasts to eliminate the contribution of endogenous fibronectin during the analysis. FN-/- fibroblasts plated on the cell-binding domain of fibronectin or on antibodies directed against mouse beta1 integrin chains attach but fail to spread and do not form focal adhesions or actin stress fibers. When such cells are treated with antibodies directed against the ectodomain of mouse syndecan-4, they spread fully and assemble focal adhesions and actin stress fibers indistinguishable from those seen in cells plated on intact fibronectin. These results identify syndecan-4 as a heparan sulfate proteoglycan involved in the assembly process. The antibody-stimulated assembly of focal adhesions and actin stress fibers in cells plated on the cell-binding domain of fibronectin can be blocked with C3 exotransferase, an inhibitor of the small GTP-binding protein Rho. Treatment of cells with lysophosphatidic acid, which activates Rho, results in full spreading and assembly of focal adhesions and actin stress fibers in fibroblasts plated on the cell-binding domain of fibronectin. We conclude that syndecan-4 and integrins can act cooperatively in generating signals for cell spreading and for the assembly of focal adhesions and actin stress fibers. We conclude further that these joint signals are regulated in a Rho-dependent manner.

Suppression of keratinocyte proliferation by plasminogen activator inhibitor-2.

We have previously shown that urokinase plasminogen activator (uPA) stimulates the growth of human keratinocytes in culture. For this effect, uPA activity is essential to generate the active amino terminal fragment, by an autolytic process. Our findings indicated further that inhibition of uPA may result in the suppression of growth of keratinoytes. Here, we provide evidence that plasminogen activator inhibitor (PAI)-2 has an anti-proliferative effect on keratinocytes. The uPA activity in cultured keratinocytes increased in parallel with cell proliferation, reaching a maximum level at confluency and decreasing gradually thereafter. The analysis of synchronized cells showed that the peak uPA activity in the medium occured just prior to S-phase, suggesting that the production and secretion of uPA is related to cell proliferation. In contrast, PAI-2 levels showed a steady increase, even after confluency. When PAI-2, purified from human cornified cells, was added to synchronized keratinocytes, S-phase was no longer evident and the peak uPA activity was eliminated. In experiments with a bacterially expressed PAI-2 fusion protein, [3H]thymidine incorporation by keratinocytes was significantly suppressed, confirming an anti-proliferative effect of PAI-2. These results strongly suggest that PAI-2 is involved in the regulation of keratinocyte proliferation and differentiation.

Mice lacking matrilin-1 (cartilage matrix protein) have alterations in type II collagen fibrillogenesis and fibril organization.

Matrilin-1 (cartilage matrix protein) is a homotrimeric protein that forms collagen-dependent and collagen-independent fibrils in the extracellular matrix of cartilage. In the growth plate of developing long bones, the gene for matrilin-1 is transcribed exclusively by the chondrocytes of the zone of maturation which is situated between the zones of proliferation and hypertrophy. When associated with the cartilage collagen fibril, which consists of collagens type II, IX, and XI, matrilin-1 displays a periodicity of 59.3 nm. Matrilin-1 also interacts with the proteoglycan, aggrecan. Because of its association with the collagen fibril, we tested the hypothesis that matrilin-1 may play a role in collagen fibril formation and cartilage matrix assembly by generating mice with targeted mutations in the matrilin-1 gene. Ultrastructural studies of the cartilage of growth plates of matrilin-1 null mice reveal an abnormal type II collagen fibrillogenesis and fibril organization in the matrix of the zone of maturation. These results represent the first report on the regulation of the heterotypic type II collagen fibril by a non-collagenous protein. The abnormal fibrillogenesis had no obvious effects on skeletal development, on the organization of chondrocytes in the growth plate and on the deposition of aggrecan and the hypertrophic-specific type X collagen in the cartilaginous matrix.

Fibroblast growth factor 2 can replace ectodermal signaling for feather development.

The initiation and morphogenesis of cutaneous appendages depend on a series of reciprocal signaling events between the epithelium and mesenchyme of the embryonic skin. In the development of feather germs, early dermal signals induce the formation of epidermal placodes that in turn signal the mesoderm to form dermal condensations immediately beneath them. We find a spatially and temporally restricted pattern of transcription for the genes that encode fibroblast growth factor (FGF) 2 and FGF receptor (FGFR) 1 in developing feather germs of the chicken embryo. FGF-2 expression is restricted to the epidermal placodes, whereas FGFR-1 expression is limited to the dermal condensations. Transcription of these genes could not be detected in skins of scaleless (sc/sc) embryos that fail to develop feathers as a result of an ectodermal defect. Treatment of sc/sc skins with FGF-2 results in the formation of feathers at the site of application of the growth factor and the induced feathers express FGFR-1 in their dermal condensations. Thus, we have established FGF-2 as an epidermal signal in early feather germ formation. The observation that FGF-2 can rescue the mutant phenotype of sc/sc embryos suggests that FGF-2 either is, or is downstream from, the signal that the sc/sc mutant ectoderm fails to generate.

Cartilage matrix protein forms a type II collagen-independent filamentous network: analysis in primary cell cultures with a retrovirus expression system.

Cartilage matrix protein (CMP) is expressed specifically in mature cartilage and consists of two von Willebrand factor A domains (CMP-A1 and CMP-A2) that are separated by an epidermal growth factor-like domain, and a coiled-coil tail domain at the carboxyl terminal end. We have shown previously that CMP interacts with type II collagen-containing fibrils in cartilage. In this study, we describe a type II collagen-independent CMP filament and we analyze the structural requirement for the formation of this type of filament. Recombinant wild-type CMP and two mutant forms were expressed in chick primary cell cultures using a retrovirus expression system. In chondrocytes, the wild-type virally encoded CMP is able to form disulfide bonded trimers and to assemble into filaments. Filaments also form with CMP whose Cys455 and Cys457 in the tail domain were mutagenized to prevent interchain disulfide bond formation. Therefore, intermolecular disulfide bonds are not necessary for the assembly of CMP into filaments. Both the wild-type and the double cysteine mutant also form filaments in fibroblasts, indicating that chondrocyte-specific factors are not required for filament formation. A truncated form of CMP that consists only of the CMP-A2 domain and the tail domain can form trimers but fails to form filaments, indicating that the deleted CMP-A1 domain and/or the epidermal growth factor domain are necessary for filament assembly but not for trimer formation. Furthermore, the expression of the virally encoded truncated CMP in chondrocyte culture disrupts endogenous CMP filament formation. Together these data suggest a role for CMP in cartilage matrix assembly by forming filamentous networks that require participation and coordination of individual domains of CMP.

Progression and recapitulation of the chondrocyte differentiation program: cartilage matrix protein is a marker for cartilage maturation.

During endochondral bone formation, chondrocytes in the cartilaginous anlage of long bones progress through a spatially and temporally regulated differentiation program before being replaced by bone. To understand this process, we have characterized the differentiation program and analyzed the relationship between chondrocytes and their extracellular environment in the regulation of the program. Our results indicate that, within an epiphyseal growth plate, the zone of proliferating chondrocytes is not contiguous with the zone of hypertrophic chondrocytes identified by the transcription of the type X collagen gene. We find that the postproliferative chondrocytes which make up the zone between the zones of proliferation and hypertrophy specifically transcribe the gene for cartilage matrix protein (CMP). This zone has been termed the zone of maturation. The identification of this unique population of chondrocytes demonstrates that the chondrocyte differentiation program consists of at least three stages. CMP translation products are present in the matrix surrounding the nonproliferative chondrocytes of both the zones of maturation and hypertrophy. Thus, CMP is a marker for postmitotic chondrocytes. As a result of the changes in gene expression during the differentiation program, chondrocytes in each zone reside in an extracellular matrix with a unique macromolecular composition. Chondrocytes in primary cell culture can proceed through the same differentiation program as they do in the cartilaginous rudiments. In culture, a wave of differentiation begins in the center of a colony and spreads to its periphery. The cessation of proliferation coincides with the appearance of CMP and eventually the cells undergo hypertrophy and synthesize type X collagen. These results reveal distinct switches at the proliferative-maturation transition and at the maturation-hypertrophy transition during chondrocyte differentiation and indicate that chondrocytes synthesize new matrix molecules and thus modify their preexisting microenvironment as differentiation progresses. However, when "terminally" differentiated hypertrophic chondrocytes are released from their surrounding environment and incubated in pellet culture, they stop type X collagen synthesis, resume proliferation, and reinitiate aggrecan synthesis. Eventually they cease proliferation and reinitiate CMP synthesis and finally type X collagen. Thus they are capable of recapitulating all three stages of the differentiation program in vitro. The data suggest a high degree of plasticity in the chondrocyte differentiation program and demonstrate that the progression and maintenance of this program is regulated, at least in part, by the extracellular environment which surrounds a differentiating chondrocyte during endochondral bone formation.

Protein kinase C regulates the recruitment of syndecan-4 into focal contacts.

Cell surface heparan sulfate proteoglycans have been implicated as co-receptors facilitating cell adhesion and growth factor binding. Recent studies on the role of a family of transmembrane heparan sulfate proteoglycans, syndecans, in cell adhesion has identified one member, syndecan-4, to be present within focal contacts. The current study investigates the mechanisms regulating the association of syndecan-4 with focal contacts based upon its immunolocalization with vinculin in quiescent, serum-stimulated, and 12-0-tetradecanoylphorbol 13-acetate (TPA)-induced cultures. In quiescent cells, syndecan-4 did not localize to focal contacts. However, activation of protein kinase C by TPA or serum induces the active recruitment of syndecan-4 into focal contacts. This induction preferentially localizes syndecan-4 to focal contacts behind the leading lamella, the subnuclear region, and along the trailing edge of migratory cells. Focal contacts in either freshly adhered cells or in the leading lamellae of migrating cells did not stain for syndecan-4. In addition to the observed subcellular distribution and recruitment, syndecan-4 was observed to co-localize with endogenously synthesized fibronectin fibrils within focal contacts as well as with fibrils present in the matrix. These findings suggest that protein kinase C activation results in syndecan-4 recruitment to focal contacts and its association with sites of matrix deposition.

The role of coiled-coil alpha-helices and disulfide bonds in the assembly and stabilization of cartilage matrix protein subunits. A mutational analysis.

Cartilage matrix protein (CMP) exists as a disulfide-bonded homotrimer in the matrix of cartilage. Each monomer consists of two CMP-A domains that are separated by an epidermal growth factor-like domain. A heptad repeat-containing tail makes up the carboxyl-terminal domain of the protein. The secreted form of CMP contains 12 cysteine residues numbered C1 through C12. Two of these are in each of the CMP-A domains, six are in the epidermal growth factor-like domain, and two are in the heptad repeat-containing tail. Two major categories of mutant CMPs were generated to analyze the oligomerization process of CMP: a mini-CMP and a heptadless full-length CMP. The mini-CMP consists of the CMP-A2 domain and the heptad repeat-containing tail. In addition, a number of mutations affecting C9 through C12 were generated within the full-length, the mini-, and the heptad-less CMPs. The mutational analysis indicates that the heptad repeats are necessary for the initiation of CMP trimerization and that the two cysteines in the heptad repeat-containing tail are both necessary and sufficient to form intermolecular disulfide bonds in either full-length or mini-CMP. The two cysteines within a CMP-A domain form an intradomain disulfide bond.

Tissue-nonspecific alkaline phosphatase participates in the establishment and growth of feather germs in embryonic chick skin cultures.

Alkaline phosphatase activity is present in the mesoderm of embryonic chick skin and becomes spatially restricted to the dermal condensation of the developing feather germs. Inhibitors to tissue-nonspecific (liver/bone/kidney), but not intestinal alkaline phosphatase inhibit the establishment and growth of feather germs in cultured skins. A window of maximum sensitivity to the inhibitor was observed to be the first day of culture when early development and establishment of pattern takes place. The cDNA for the avian tissue-nonspecific alkaline phosphatase was cloned and sequenced, and Southern analysis revealed a single copy of this gene in the avian genome. Northern analysis revealed that a 2.8 kb transcript for this form of alkaline phosphatase is present in developing skin.

Biotinylated hyaluronic acid as a probe for identifying hyaluronic acid-binding proteins.

The glycosaminoglycans hyaluronan (HA), heparin, and chondroitin sulfate were biotinylated using biotin-x-hydrazide (biotin-epsilon-aminocaproyl hydrozyde) in conjunction with N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride, an activating agent for carboxyl groups. The biotin-x-hydrazide was shown to be coupled directly to the glycosaminoglycans in enzymatic digestions and competition experiments. The biotinylated HA was shown to bind to link protein and receptor for hyaluronic acid-mediated motility, two proteins known to bind HA. The labeled HA was used as a probe to detect known HA-binding proteins in chicken cartilage extract and to identify new HA-binding motifs in the G3 domain of the proteoglycan aggrecan. The significance of the biotinylation of HA, heparin, and chondroitin sulfate A are discussed.