The effect of dietary protein deficiency on albumin synthesis and on the concentration of active albumin messenger ribonucleic acid in rat liver.

After severe hepatic injury induced by dimethylnitrosamine, approximately a 4-fold increase in hepatic prolyl 4-hydroxylase activity occurred within 4 days, whereas the increases in total immunoreactive prolyl 4-hydroxylase protein and in prolyl 3-hydroxylase activity were only about 1.4-fold. The different magnitudes of the increases in the prolyl 4-hydroxylase and 3-hydroxylase activities were verified after partial purification of the enzymes by gel filtration. The data support previous reports indicating differential increases in the activities of individual enzymes of collagen biosynthesis in hepatic injury. Separation of prolyl 4-hydroxylase tetramers from the monomer-size protein by gel filtration indicated that the increase in enzyme activity was similar to that in enzyme tetramers, and an increase had also occurred in the ratio of enzyme tetramers to total enzyme protein. Thus the specific activity of the tetramers had remained unchanged in liver injury. The administration of dimethylnitrosamine was also accompanied by a marked increase in the immunoreactive prolyl 4-hydroxylase protein concentration in the serum, and a similar effect was also noted after carbon tetrachloride administration, results suggesting that the increases originated in the liver.     

Effect of prevention of procollagen triple-helix formation on proline 3-hydroxylation in freshly isolated chick-embryo tendon cells.

Inhibition of procollagen triple-helix formation by the addition of cis-hydroxyproline or azetidine-2-carboxylic acid increased the synthesis of 3-hydroxy[14C]proline 1.7-1.8-fold in pulse-chase experiments with freshly isolated chick-embryo tendon cells. The amount of 3-hydroxy[14C]proline, expressed as a percentage of the total 14C radioactivity in hydroxyproline, reached 8.4%. Control experiments indicated that the two analogues had no effect on the prolyl 3-hydroxylase activity of these cells. The data suggest that the time available before triple-helix formation in part regulates the extent of the 3-hydroxylation of proline in the biosynthesis of collagen in intact cells.     

Stuck on you: Meninges cellular crosstalk in development

The spatial and temporal development of the brain, overlying meninges (fibroblasts, vasculature and immune cells) and calvarium are highly coordinated. In particular, the timing of meningeal fibroblasts into molecularly distinct pia, arachnoid and dura subtypes coincides with key developmental events in the brain and calvarium. Further, the meninges are positioned to influence development of adjacent structures and do so via depositing basement membrane and producing molecular cues to regulate brain and calvarial development. Here, we review the current knowledge of how meninges development aligns with events in the brain and calvarium and meningeal fibroblast “crosstalk” with these structures. We summarize outstanding questions and how the use of non-mammalian models to study the meninges will substantially advance the field of meninges biology.     

Regulation of proline 3-hydroxylation and prolyl 3-hydroxylase and 4-hydroxylase activities in transformed cells.

Prolyl 3-hydroxylase activity and the extent of collagen proline 3-hydroxylation were studied in six transformed and three control human cell lines. In the transformed cell lines, the enzyme activity was markedly high in two, similar to that in control cells in two and significantly low in two. The extent of proline 3-hydroxylation was markedly high in cell lines with high enzyme activity, but it was also significantly high in some transformed cell lines with enzyme activities similar to those in the controls. The results thus suggest that, in addition to the amount of enzyme activity present, the rate of collagen synthesis also affects the extent of proline 3-hydroxylation in the newly synthesized collagen. The effect of acute cell transformation on prolyl 3-hydroxylase and 4-hydroxylase activities was studied by infecting chick-embryo fibroblasts with Rous sarcoma virus mutant NY68, temperature-sensitive for transformation. At the permissive temperature prolyl 3-hydroxylase activity showed a more rapid increase and decrease than did prolyl 4-hydroxylase activity, the maximal activity for both enzymes being about 2.5 times that in the control chick fibroblasts. When the transformed cells were shifted to the non-permissive temperature the decays in the elevated enzyme activities were similar, suggesting identical half-lives.     

Correlation of the steady-state RNA levels among the alpha-subunit of prolyl 4-hydroxylase and the alpha 1 and alpha 2 chains of type I collagen during growth of chicken embryo tendon fibroblasts.

The relative steady-state levels of RNAs encoding type I collagen and prolyl 4-hydroxylase were examined in exponentially growing primary cultures of chicken embryo tendon fibroblasts. The RNA levels of the alpha 1 and alpha 2 chains of type I collagen were maximal when the fibroblasts reached the confluent state. The RNA levels of the alpha-subunit of prolyl 4-hydroxylase were also maximal at confluency and rose and fell with the RNA levels of the two collagen chains. The RNA levels of the beta-subunit of prolyl 4-hydroxylase did not correlate with the changes observed for the alpha-subunit or for either chain of type I collagen. The RNA levels of the beta-subunit were slightly higher than the RNA levels of the alpha-subunit. These results support our hypothesis that the synthesis of the alpha-subunit and thus the association of newly synthesized alpha-subunits with pre-existing beta-subunits is the rate-limiting factor in determining prolyl 4-hydroxylase activity in cultured cells.     

Developmental biology of the meninges

The meninges are membranous layers surrounding the central nervous system. In the head, the meninges lie between the brain and the skull, and interact closely with both during development. The cranial meninges originate from a mesenchymal sheath on the surface of the developing brain, called primary meninx, and undergo differentiation into three layers with distinct histological characteristics: the dura mater, the arachnoid mater, and the pia mater. While genetic regulation of meningeal development is still poorly understood, mouse mutants and other models with meningeal defects have demonstrated the importance of the meninges to normal development of the calvaria and the brain. For the calvaria, the interactions with the meninges are necessary for the progression of calvarial osteogenesis during early development. In later stages, the meninges control the patterning of the skull and the fate of the sutures. For the brain, the meninges regulate diverse processes including cell survival, cell migration, generation of neurons from progenitors, and vascularization. Also, the meninges serve as a stem cell niche for the brain in the postnatal life. Given these important roles of the meninges, further investigation into the molecular mechanisms underlying meningeal development can provide novel insights into the coordinated development of the head.     

Hypoxic enhancement of type IV collagen secretion accelerates adipose conversion of 3T3-L1 fibroblasts

Hypoxic modulation of collagen metabolism appears to be related to pathogenesis of many diseases such as fibrosis of connective tissue after injury and scleroderma. Since most of our understanding of how procollagen assembles within the cell has come from studies on cells cultured under normoxia, it may not be helpful for the etiology of the diseases observed in peripheral tissues under hypoxic conditions. As an experimental model for the hypoxic modulation of collagen metabolism, we cultured 3T3-L1 fibroblasts under low partial oxygen pressure and found that hypoxia enhances secretion of type IV collagen 10-fold and accelerates adipose conversion of the cells. The enhanced secretion of type IV collagen was not accompanied by an appreciable increase of alpha1(IV) and alpha2(IV) mRNAs. Prolyl 4-hydroxylase alpha increased only 3-fold under hypoxia. We suggest that hypoxia creates an environment of prolyl 4-hydroxylase alpha(2)beta(2) tetramers favorable for the folding of type IV procollagen which has many interruptions of the Gly-Xaa-Yaa repeat.     

Ultrastructural localization of the membrane-bound Mg-adenosine triphosphatase activity in rat meninges

The distribution of the membrane-bound magnesium ions-dependent adenosine triphosphatase (Mg-ATPase) activity has been studied ultracytochemically in rat meninges by the method of Wachstein and Meisel (1957). A device specially constructed to avoid preparation artefacts has been used to obtain sections from the parietal region of the head. The meninges display an intense though irregularly distributed ATPase activity marked by depositions of electron-dense reaction product (RP) which is almost absent in the outer and middle dural layers. In the borderline zone between dura mater and the arachnoid the RP deposits are found at the outer surface of the inner dural cells and at the contact sites between these cells and the dural neurothelium. The intercellular cleft(s) between the neurothelium and the outer arachnoidal layer, occupied by an "electron-dense band", remains free of RP. The strongest accumulations of reactions granules are observed on the surface of the leptomeningeal cells of the arachnoidal space. In the contact region between the inner arachnoidal and the outer pial layers the distribution of the RP is similar to the one observed in the interface zone dura mater/arachnoid, while the pial cells themselves are definitely reaction-positive. In all meningeal vessels RP is found at the lumenal and abluminal aspects of the endothelium as well as at the cell membranes of the perivascular cells. These results emphasize the importance of the dural neurothelium for the functions of the blood-cerebrospinal fluid (CSF)-barrier between the dural blood vessels and the CSF.     

Prolyl 3-hydroxylase 1, enzyme characterization and identification of a novel family of enzymes

The collagen prolyl hydroxylases are enzymes that are required for proper collagen biosynthesis, folding, and assembly. They reside within the endoplasmic reticulum and belong to the group of 2-oxoglutarate and iron-dependent dioxygenases. Although prolyl 4-hydroxylase has been characterized as an alpha2beta2 tetramer in which protein disulfide isomerase is the beta subunit with two different alpha subunit isoforms, little is known about the enzyme prolyl 3-hydroxylase (P3H). It was initially characterized and shown to have an enzymatic activity distinct from that of prolyl 4-hydroxylase, but no amino acid sequences or genes were ever reported for the mammalian enzyme. Here we report the characterization of a novel prolyl 3-hydroxylase enzyme isolated from embryonic chicks. The primary structure of the enzyme, which we now call P3H1, demonstrates that P3H1 is a member of a family of prolyl 3-hydroxylases, which share the conserved residues present in the active site of prolyl 4-hydroxylase and lysyl hydroxylase. P3H1 is the chick homologue of mammalian leprecan or growth suppressor 1. Two other P3H family members are the genes previously called MLAT4 and GRCB. In this study we demonstrate prolyl 3-hydroxylase activity of the purified enzyme P3H1 on a full-length procollagen substrate. We also show it to specifically interact with denatured collagen and to exist in a tight complex with other endoplasmic reticulum-resident proteins. Immunohistochemistry with a monoclonal antibody specific for chick P3H1 localizes P3H1 specifically to tissues that express fibrillar collagens, suggesting that other P3H family members may be responsible for modifying basement membrane collagens.     

Increases in mRNA concentrations of the alpha and beta subunits of prolyl 4-hydroxylase accompany increased gene expression of type IV collagen during differentiation of mouse F9 cells.

Mouse F9 teratocarcinoma stem cells differentiate in monolayer cultures in the presence of retinoic acid, dibutyryl cAMP, and isobutyl methylxanthine. This differentiation is associated with a marked increase in the synthesis rates and mRNA concentrations of basement membrane proteins such as type IV collagen. We report here that the differentiation also involves an increase of up to 50-fold in the concentrations of the mRNAs for the alpha and beta subunits of prolyl 4-hydroxylase, the enzyme required for the cotranslational and post-translational hydroxylation of proline residues in collagens. The time courses and magnitudes of increases in these two mRNA concentrations were similar to those observed in the same experiments for the mRNA of the alpha chain of type IV collagen. In the differentiated F9 cells the concentration of the alpha subunit mRNA was about 30% of the beta subunit mRNA concentration. Northern blot analyses indicated that the sizes of the alpha and beta subunit mRNAs in the differentiated mouse F9 cells are similar to those in human skin fibroblasts. The F9 cell differentiation system appears to provide a useful model for studies on the regulation of prolyl 4-hydroxylase synthesis.     

Morphogenesis of rat cranial meninges

Summary The meninges of albino Wistar rat embryos, aged between the 11th embryonic day (ED) and birth, were sectioned using a specially constructed device. This technique permits optimal microanatomical preservation of all tissues covering the convexity of the brain: skin, muscle, cartilage or bone, and the meninges. At ED11, the zone situated between the epidermis and the brain is occupied by a mesenchymal network. At ED12, part of this delicate network develops as a dense outer cellular layer, while the remainder retains its reticular appearance, thus forming an inner layer (the future meningeal tissue). At ED13, the dura mater starts to differentiate. At ED14, the bony anlage of the skull can be identified, and along with the proceeding maturation of dura mater some fibrillar structures resembling skeletal muscle fibers appear in the developing arachnoid space. At ED15–17, a primitive interface zone — dura mater/ arachnoid — is formed, comprised by an outer electronlucent and an inner electron-dense layer marking the outer aspect of the arachnoidal space. At ED18–19, the innermost cellular row of the inner durai layer transforms into neurothelium, which is separated from the darker arachnoidal cells by an electron-dense band. The arachnoidal trabecular zone with the leptomeningeal cells is formed at ED19. By the end of the prenatal period (ED20–21), its innermost part organizes into an inner arachnoidal layer and an outer and inner pial layer. The results from this study indicate (i) that dura mater and leptomeninges develop from an embryonic network of connective tissue-forming cells, and (ii) that the formation of cerebrospinal fluid (CSF)-containing spaces accompanies the differentiation of the meningeal cellular layers.     

Intravital Imaging of a Massive Lymphocyte Response in the Cortical Dura of Mice after Peripheral Infection by Trypanosomes

Peripheral infection by Trypanosoma brucei, the protozoan responsible for sleeping sickness, activates lymphocytes, and, at later stages, causes meningoencephalitis. We have videoed the cortical meninges and superficial parenchyma of C56BL/6 reporter mice infected with T.b.brucei. By use of a two-photon microscope to image through the thinned skull, the integrity of the tissues was maintained. We observed a 47-fold increase in CD2+ T cells in the meninges by 12 days post infection (dpi). CD11c+ dendritic cells also increased, and extravascular trypanosomes, made visible either by expression of a fluorescent protein, or by intravenous injection of furamidine, appeared. The likelihood that invasion will spread from the meninges to the parenchyma will depend strongly on whether the trypanosomes are below the arachnoid membrane, or above it, in the dura. Making use of optical signals from the skull bone, blood vessels and dural cells, we conclude that up to 40 dpi, the extravascular trypanosomes were essentially confined to the dura, as were the great majority of the T cells. Inhibition of T cell activation by intraperitoneal injection of abatacept reduced the numbers of meningeal T cells at 12 dpi and their mean speed fell from 11.64 ± 0.34 μm/min (mean ± SEM) to 5.2 ± 1.2 μm/min (p = 0.007). The T cells occasionally made contact lasting tens of minutes with dendritic cells, indicative of antigen presentation. The population and motility of the trypanosomes tended to decline after about 30 dpi. We suggest that the lymphocyte infiltration of the meninges may later contribute to encephalitis, but have no evidence that the dural trypanosomes invade the parenchyma.     

Liver collagen hydroxylation in murine schistosomiasis.

The activity of procollagen prolyl hydroxylase was measured in fibrotic liver obtained from mice with hepatosplenic schistosomiasis, an animal model of the most prevalent form of human liver fibrosis. Measurable activity of prolyl hydroxylase in fibrotic liver supernatants was 47-fold higher than that of normal liver. The effect of prolyl hydroxylase inhibition on collagen synthesis in fibrotic liver slices was studied, using 8,9-dihydroxy-7-methyl benzo[b]quinolizinium bromide (GPA 1734). This compound was shown in other systems to inhibit prolyl and lysyl hydroxylations by iron chelation at concentrations which did not affect total protein synthesis. The formation of nondialyzable labelled hydroxyproline was inhibited by GPA 1734, 40, 70 and 95% at 30, 50 and 100 micrometer, respectively. Incorporation of proline into total liver protein was unaffected at 30 and 50 micrometer, but was inhibited 20% at 100 micrometer GPA 1734. Underhydroxylated collagen synthesized by liver slices with GPA 1734 was extracted with neutral salt solution and was subsequently hydroxylated with partially-purified prolyl hydroxylase to the same extent as control material synthesized in the absence of GPA 1734.     

Reduction of collagen production in keloid fibroblast cultures by ethyl-3,4-dihydroxybenzoate. Inhibition of prolyl hydroxylase activity as a mechanism of action

Excessive accumulation of collagen is the hallmark of several clinical conditions characterized by tissue fibrosis. Previously, 3,4-dihydroxybenzoic acid, a structural analog of alpha-ketoglutarate and ascorbate, has been shown to inhibit the activity of purified prolyl 4-hydroxylase, the enzyme catalyzing the synthesis of 4-hydroxyproline during intracellular biosynthesis of procollagen. In this study a hydrophobic modification, an ethyl ester, of 3,4-dihydroxybenzoic acid was tested for its effects on collagen synthesis and prolyl hydroxylase activity in human skin fibroblast cultures. The results indicated that 0.4 mM ethyl-3,4-dihydroxybenzoate markedly inhibited the synthesis of 4-hydroxyproline in normal cell cultures apparently as a result of reduced prolyl 4-hydroxylase activity, and the synthesis and secretion of both type I and type III procollagens were markedly reduced. Control experiments indicated that the test compound did not affect the viability, proliferation, or plating efficiency of the cells, and it had little, if any, effect on the synthesis of noncollagenous proteins. Furthermore, determinations of type I and type III procollagen mRNA steady-state levels by slot-blot hybridizations suggested that the inhibition of procollagen production did not occur on the pretranslational level. Thus, ethyl-3,4-dihydroxybenzoate selectively reduced procollagen production in fibroblast cultures by inhibiting the post-translational synthesis of 4-hydroxyproline. Similar inhibition was also observed in keloid fibroblast cultures, demonstrating the potential applicability of ethyl-3,4-dihydroxybenzoate, or other structural alpha-ketoglutarate or ascorbate analogs, for treatment of fibrotic diseases.     

Studying Human Brain Inflammation in Leptomeningeal and Choroid Plexus Explant Cultures

The meninges (dura, pia and arachnoid) are critical membranes encasing and protecting the brain within the skull. The leptomeninges, which comprise the arachnoid and pia, have many functions beyond brain protection including roles in neurogenesis, fibrotic scar formation and brain inflammation. Similarly, the choroid plexus plays important roles in normal brain function but is also involved in brain inflammation. We have begun studying the role of human leptomeninges and choroid plexus in brain inflammation and leptomeninges in fibrotic scar formation, using human brain derived explant cultures. To study the composition of the cells generated in these explants we undertook immunocytochemical characterisation. Cells, mainly pericytes and meningeal macrophages, emerge from leptomeningeal explants (LME’s) and respond to inflammatory mediators by producing inflammatory molecules. LME-derived cells also respond to mechanical injury and cytokines, providing an in vitro human brain model of fibrotic scar formation. Choroid plexus explants (CPE’s) generate epithelial cells, pericytes and microglia/macrophages. CPE-derived cells also respond to inflammatory mediators. LME and CPE explants survive and generate cells for many months in vitro and provide a remarkable opportunity to study basic mechanisms of human brain inflammation and fibrosis and to test human-active anti-inflammatory and anti-scarring treatments.     

Macrophages and dendritic cells in the rat meninges and choroid plexus: three-dimensional localisation by environmental scanning electron microscopy and confocal microscopy

The present investigation provides novel information on the topographical distribution of macrophages and dendritic cells (DCs) in normal meninges and choroid plexus of the rat central nervous system (CNS). Whole-mounts of meninges and choroid plexus of Lewis rats were incubated with various anti-leucocyte monoclonal antibodies and either visualised with gold-conjugated secondary antibody followed by silver enhancement and subsequent examination by environmental scanning electron microscopy or by the use of fluorochromes and confocal microscopy. Large numbers of MHC class II⁺ putative DCs were identified on the internal or subarachnoid aspect of dural whole-mounts, on the surface of the cortex (pia/arachnoid) and on the surface of the choroid plexus. Occupation of these sites would allow DCs access to cerebrospinal fluid (CSF) and therefore allow antigens into the subarachnoid space and ventricles. By contrast, macrophages were less evident at sites exposed to CSF and were more frequently located within the connective tissue of the dura/arachnoid and choroid plexus stroma and also in a sub-pial location. The present data suggest that DC may be strategically located within the CNS to sample CSF-borne antigens. Furthermore, the data suggest that CNS tissue samples collected without careful removal of the meninges may inadvertantly be contaminated by DCs and meningeal macrophages.     

Liver collagen hydroxylation in murine schistosomiasis

The activity of procollagen prolyl hydroxylase was measured in fibrotic liver obtained from mice with hepatosplenic schistosomiasis, an animal model of the most prevalent form of human liver fibrosis. Measurable activity of prolyl hydroxylase in fibrotic liver supernatants was 47-fold higher than that of normal liver.The effect of prolyl hydroxylase inhibition on collagen synthesis in fibrotic liver slices was studied, using 8,9-dihydroxy-7-methyl benzo[b]quinolizinium bromide (GPA 1734). This compound was shown in other systems to inhibit prolyl and lysyl hydroxylations by iron chelation at concentrations which did not affect total protein synthesis. The formation of nondialyzable labelled hydroxyproline was inhibited by GPA 1734, 40, 70 and 95% at 30, 50 and 100 μM, respectively. Incorporation of proline into total liver protein was unaffected at 30 and 50 μM, but was inhibited 20% at 100μM GPA 1734. Underhydroxylated collagen synthesized by liver slices with GPA 1734 was extracted with neutral salt solution and was subsequently hydroxylated with partially-purified prolyl hydroxylase to the same extent as control material synthesized in the absence of GPA 1734.     

Lack of collagen type specificity for lysyl hydroxylase isoforms

Lysyl hydroxylase is the enzyme catalyzing the formation of hydroxylysyl residues in collagens. Large differences in the extent of hydroxylysyl residues are found among collagen types. Three lysyl hydroxylase isoenzymes (LH1, LH2, LH3) have recently been characterized from human and mouse tissues. Nothing is known about the distribution of these isoforms within cells or whether they exhibit collagen type specificity. We measured mRNA levels of the three isoforms, as well as the mRNAs of the main collagen types I, III, IV, and V and the alpha subunit of prolyl 4-hydroxylase, another enzyme involved in collagen biosynthesis, in different human cell lines. Large variations were found in mRNA expression of LH1 and LH2 but not LH3. Immunoblotting was utilized to confirm the results of Northern hybridization. The levels of mRNA of LH1, LH2, and the alpha subunit of prolyl 4-hydroxylase showed significant correlations with each other. The LH3 mRNA levels did not correlate with those of LH1, LH2, or the alpa subunit of prolyl 4-hydroxylase, clearly indicating a difference in the regulation of LH3. No correlation was observed between LH isoforms and individual collagen types, indicating a lack of collagen type specificity for lysyl hydroxylase isoforms. Our observations suggest that LH1, LH2, and the alpha subunit of prolyl 4-hydroxylase are coregulated together with total collagen synthesis but not with the specific collagen types and indicate that LH3 behaves differently from LH1 and LH2, implying a difference in their substrates. These observations set the basis for further studies to define the functions of lysyl hydroxylase isoforms.