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In order to understand the pathobiology of neurotrophic keratopathy, we established a mouse model by coagulating the first branch of the trigeminal nerve (V1 nerve). In our model, the sensory nerve in the central cornea disappeared and remaining fibers were sparse in the peripheral limbal region. Impaired corneal epithelial healing in the mouse model was associated with suppression of both cell proliferation and expression of stem cell markers in peripheral/limbal epithelium as well as a reduction of transient receptor potential vanilloid 4 (TRPV4) expression in tissue. TRPV4 gene knockout also suppressed epithelial repair in mouse cornea, although it did not seem to directly modulate migration of epithelium. In a co-culture experiment, TRPV4-introduced KO trigeminal ganglion upregulated nerve growth factor (NGF) in cultured corneal epithelial cells, but ganglion with a control vector did not. TRPV4 gene introduction into a damaged V1 nerve rescues the impairment of epithelial healing in association with partial recovery of the stem/progenitor cell markers and upregulation of cell proliferation and of NGF expression in the peripheral/limbal epithelium. Gene transfer of TRPV4 did not accelerate the regeneration of nerve fibers. Sensory nerve TRPV4 is critical to maintain stemness of peripheral/limbal basal cells, and is one of the major mechanisms of homeostasis maintenance of corneal epithelium.

The present study attempts to elucidate the role of TRPV1 cation channel receptor on primary repair in an incision-wounded mouse cornea in vivo. Previous study revealed that blocking TRPV1 suppressed myofibroblast formation and expression of transforming growth factor β1 (TGFβ1) in cultured keratocytes or ocular fibroblasts. Male C57BL/6 (wild-type; WT) mice and male C57BL/6 Trpv1-null (KO) mice incurred a full-thickness incision injury (1.8 mm in length, limbus to limbus) in the central cornea of one eye with a surgical blade under general and topical anesthesia. The injury was not sutured. On days 0, 5, and 10, the eyes were enucleated, processed for histology, immunohistochemistry, and real-time RT-PCR gene expression analysis to evaluate the effects of the loss of TRPV1 on primary healing. Electron microscopy observation was also performed to know the effect of the loss of TRPV1 on ultrastructure of keratocytes. The results showed that the loss of Trpv1 gene delayed closure of corneal stromal incision with hindered myofibroblast transdifferentiation along with declines in the expression of collagen Ia1 and TGFβ1. Inflammatory cell infiltration was not affected by the loss of TRPV1. Ultrastructurally endoplasmic reticulum of TRPV1-null keratocytes was more extensively dilated as compared with WT keratocytes, suggesting an impairment of protein secretion by TRPV1-gene knockout. These results indicate that injury-related TRPV1 signal is involved in healing of stromal incision injury in a mouse cornea by selectively stimulating TGFβ-induced granulation tissue formation.

Background and purpose Meibomian glands (MGs) play an important role in the maintenance of ocular surface health, but the mechanisms of their development are still poorly understood. The MGs arise from the epithelium at the junction of eyelid fusion, raising the possibility that defective eyelid fusion disturbs the formation of MGs. Methods We examined, histologically and functionally, the development of MGs in mice with either normal or defective eyelid fusion, displaying eye-closed at birth (ECB) or eye-open at birth (EOB) phenotypes, respectively. Results The Meibomian anlage was detected in the epithelium at the eyelid fusion junction immediately after birth at postnatal day 0 (PD0), and it extended into the eyelid stroma at PD1 and started to branch and produce meibum at PD7 in the ECB mice. In contrast, few if any MG structures were detectable in the EOB mice in the early postnatal periods. The Meibomian gland ductile system was seen aligned along the eyelid margin in the adult ECB mice, but was absent or scarce in that of the EOB mice. While MG abnormalities were found in all EOB mice, the severity varied and corresponded to the position and the size of eye opening but not the genetic defects of the mice. Conclusion Proper Meibomian gland formation and development require eyelid closure and fusion.

Using differential salt fractionation, different collagen types were obtained from corneas of 17-day-old chick embryos. The collagen precipitated by 1.7 M and 2.5 M NaCl consisted of type I collagen, having an alpha 1: alpha 2 ratio of 2. The collagen precipitated by 5 M NaCl contained alpha A, alpha B, alpha 1 and alpha 2 chains having an alpha A: alpha B ratio of 1 and an alpha 1: alpha 2 ratio of 10. The alpha 1 and alpha 2 chains isolated from corneas were found to have higher mobility in polyacrylamide gel electrophoresis in sodium dodecylsulfate as compared with that of their counterparts isolated from embryonic chick tendons. When the alpha 1 and alpha 2 chains isolated by polyacrylamide gel electrophoresis in sodium dodecylsulfate were subjected to limited protease degradations, the peptide maps obtained from corneal alpha chains were not identical to those of tendon alpha chains. It is suggested that the type I collagen in cornea is different from type I collagen in tendon. Also the results indicated that the alpha 1 chain present in the 5 M NaCl precipitate is a type I alpha 1 chain.

Tubedown-1 (tbdn-1) is a mammalian homologue of the N-terminal acetyltransferase subunit NAT1 of Saccharomyces cerevisiae and copurifies with an acetyltransferase activity. Tbdn-1 expression in endothelial cells becomes downregulated during the formation of capillary-like structures in vitro and is regulated in vivo in a manner which suggests a functional role in dampening blood vessel development. Here we show that tbdn-1 is expressed highly in the vitreal vascular network (tunica vasculosa lentis and vasa hyaloidea propria) during the pruning and remodeling phases of this transient structure. The vitreal blood vessels of mice harboring a targeted inactivation of TGF-beta2 fail to remodel and abnormally accumulate, a phenomenon reminiscent of the ocular pathology resembling persistent fetal vasculature (PFV) in humans. Since suppression of normal tbdn-1 expression has been previously observed in retinal vessel proliferation, we analyzed vitreal vascular changes and tbdn-1 expression in TGF-beta2(-/-) eyes. The nuclei of vitreal vessel endothelial cells in TGF-beta2(-/-) eyes express proliferating cell nuclear antigen (PCNA) and exhibit increased levels of active (P42/44)mitogen-activated protein kinase (phospho-(P42/44)MAPK), characteristics consistent with proliferative endothelial cells. In contrast to normal vitreal vessels, collagen IV expression exhibited a disorganized pattern in the TGF-beta2(-/-) vitreal vessels, suggesting vessel disorganization and possibly a breakdown of vessel basal laminae. Moreover, vitreal vessels of TGF-beta2(-/-) mice lack expression of pericyte markers (CD13, alpha smooth muscle actin) and show ultrastructural changes consistent with pericyte degeneration. The accumulating vitreal blood vessels of TGF-beta2(-/-) mice, while maintaining expression of the endothelial marker von Willebrand Factor, show a significant decrease in the expression of tbdn-1. We addressed the functional role of tbdn-1 in the regulation of vitreal blood vessels using an in vitro model of choroid-retina capillary outgrowth. Clones of the RF/6A fetal choroid-retina endothelial cell line showing suppression of tbdn-1 levels after overexpression of an antisense TBDN-1 cDNA display a significant increase in the formation of capillary-like structures in vitro compared with controls. These findings suggest that tbdn-1 inhibits capillary-like formation in vitro and may serve to dampen vitreal blood vessel formation preceding the regression of the vitreal vasculature during development. Our results also suggest that tbdn-1 may participate with TGF-beta2 in regulating normal development of the vitreal vasculature.

To review studies of the roles of extracellular matrix (ECM) metabolism in corneal epithelium during wound repair. Methods. 1) Alterations in the structure and composition of epithelial basement membrane during corneal epithelial healing were examined histologically and immunohistochemically. 2) The effects of procollagen and hyaluronan synthesis inhibitors on the spread of rabbit corneal epithelium were determined in organ culture. 3) Expression of keratan sulfate proteoglycan (KSPG) proteins in corneal epithelium was examined during repair after injury in wild-type and lumican-null mice.1) Corneal epithelial basement membrane was transiently degraded and reassembled during tissue repair. Patterns of type IV collagen immunoreactivity were also transiently altered. The system of matrix metalloproteinase-tissue inhibitors of metalloproteinases may play an important role in the disassembly and reorganization of epithelial basement membrane. 2) Inhibitors of procollagen secretion and hyaluronan biosynthesis disrupted the spread of a corneal epithelial sheet in situ. 3) Among the corneal KSPG proteins examined, lumican was transiently expressed in migrating murine corneal epithelial cells. Anti-lumican antibody inhibited corneal epithelial resurfacing in organ culture. The absence of lumican was found to delay corneal epithelial wound healing in mice.Extracellular matrix metabolism by the injured corneal epithelium is important in the repair process.

Lumican, an extracellular matrix proteoglycan was previously shown to be upregulated with increasing severity of nonalcoholic steatohepatitis (NASH). Although lumican is involved in collagen fibrillogenesis in extra-hepatic tissues, little is known about the role of lumican in hepatic disease. We therefore determined lumican expression in etiologies other than clinical NASH. Our results indicated that lumican is upregulated in clinical samples of hepatitis C virus infection, in experimental rodent models of chronic and acute liver injury and could additionally be induced in vitro in response to the pro-fibrotic cytokine transforming growth factor β1 (TGFβ1) and to lipotoxic palmitic acid. Together, these results suggested a role for lumican in hepatic fibrosis. To investigate the functional role of lumican in hepatic fibrosis, lumican null (Null) and wild-type (WT) littermates were administered carbon tetrachloride intra-peritoneally. Serum and liver tissue were analyzed for indices of liver injury, fibrosis, matrix turnover, and proliferation. Hepatic fibrosis was greatly reduced in null animals (P

The conjunctival epithelium is intrinsically different from the corneal epithelium in vivo, but sometimes can transform into an epithelium morphologically indistinguishable from the latter after healing of a total corneal epithelial defect. It remains unclear whether this morphologic transformation represents a process of extrinsic modultation or transdifferentiation of intrinsically divergent epithelium. In air-lifted organotypic cultures, rabbit conjunctival epithelial cells lost goblet cell differentiation and were stratified to the same extent as corneal epithelial cells, resembling the above in vivo morphologic transformation. Paired expression of K3 (64 kD) and K12 (55 kD) keratins has been regarded as a marker for corneal-type differentiation. Immunoblot analysis by monoclonal antibody AE5 revealed that K3 keratin was expressed by both submerged or air-lifted corneal and conjunctival cultures with or without 3T3 fibroblasts in collagen gel. In contrast, K12 keratin was expressed only by air-lifted corneal cultures with 3T3 fibroblasts using monoclonal antibody AK2 and two epitope-specific antibodies to N- and C- terminal oligopeptides deduced from the mouse K12 gene. This finding was also confirmed by Northern hybridization with a rabbit K12 cDNA probe. The expression of K12 keratin was more delayed than that of K3 keratin in air-lifted corneal cultures. This dissociated expression of these two keratins resembles that noted in vivo in the stem cell-containing limbal region. These results suggest that morphologic transformation of the conjunctival epithelium represents extrinsic environment modulation, and that differential expression of K12 but not K3 keratin signifies corneal epithelial differentiation.

Lumican regulates collagenous matrix assembly as a keratan sulfate proteoglycan in the cornea and is also present in the connective tissues of other organs and embryonic corneal stroma as a glycoprotein. In normal unwounded cornea, lumican is expressed by stromal keratocytes. Interestingly, injured mouse corneal epithelium ectopically and transiently expresses lumican during the early phase of wound healing, suggesting a potential lumican functionality unrelated to regulation of collagen fibrillogenesis, e.g., modulation of epithelial cell adhesion or migration. Healing of a corneal epithelial injury in lumican knockout (Lum(-/-)) mice was significantly delayed compared with Lum(+/-) mice. Addition of purified lumican to cultured medium promoted re-epithelialization and enhanced cell proliferation of wild-type mouse corneal epithelial cells in an organ culture. Therefore, administration of lumican may be beneficial for treating epithelial defects in the cornea and other tissues.

Precise coordination of progenitor cell proliferation and differentiation is essential for proper organ morphogenesis and function during mammalian development. The mitogen-activated protein kinase kinase kinase 1 (MAP3K1) has a well-established role in anterior eyelid development, as Map3k1-knockout mice have defective embryonic eyelid closure and an `eye-open at birth' (EOB) phenotype. Here, we show that MAP3K1 is highly expressed in the posterior of the developing eye and is required for retina development. The MAP3K1-deficient mice exhibit increased proliferation and apoptosis, and Müller glial cell overproduction in the developing retinas. Consequently, the retinas of these mice show localized rosette-like arrangements in the outer nuclear layer, and develop abnormal vascularization, broken down retinal pigment epithelium, photoreceptor loss and early onset of retinal degeneration. Although the retinal defect is associated with increased cyclin D1 and CDK4/6 expression, and RB phosphorylation and E2F-target gene upregulation, it is independent of the EOB phenotype and of JNK. The retinal developmental defect still occurs in knockout mice that have undergone tarsorrhaphy, but is absent in compound mutant Map3k1+/ΔKDJnk1–/– and Map3k1+/ΔKDJnk+/–Jnk2+/– mice that have EOB and reduced JNK signaling. Our results unveil a novel role for MAP3K1 in which it crosstalks with the cell cycle regulatory pathways in the prevention of retina malformation and degeneration.

Many transgenic and knock-out mice exhibit pathogenesis resembling human ocular surface diseases. Thus, the clinical manifestations of mouse lines can be used as clues for identifying inherited human ocular diseases of unknown etiology. However, modified mouse lines using conventional techniques of transgenesis (insertion of an exogenesis gene into an organism such that the gene is transmitted to the offspring) and gene-targeting techniques often exhibit embryonic lethality and congenital defects precluding the use of such mouse models to study acquired ocular surface tissue diseases. Thus, it is vital to have ocular-surface tissue-specific promoters to create mouse models for studying the consequences of loss/gain of a function through transgenesis and gene-targeting techniques. We have identified two cornea-specific genes, keratin 12 (Krt12) and keratocan (Kera), and a Pax6 promoter containing regulatory elements of ocular-surface epithelium-specific expression. We have developed three mouse lines using: (1) keratocan promoter for the preparation of mice in which the promoter is active in ocular surface mesenchymal cells of neural crest origin; (2) knock-in strategies for the Krt12 promoter; and (3) Pax6 promoter to generate mice in which the promoter is active in ocular surface epithelium, that is, cornea, conjunctiva, and lacrimal and meibomian glands. These mouse lines have been bred to obtain double and triple transgenic mice that have inducible gain/loss of functions in corneal keratocyte, corneal epithelium, and ocular-surface epithelium-specific manners. Use of these mouse lines has revealed that excess fibroblast growth factor 7 causes ocular surface squamous neoplasia and the ablation of transforming growth factor-β type II receptor impairs healing of epithelium debridement by delayed activation of p38 mitogen-activated protein kinase. These strategies are of great value to examine the effects of gain and/or loss of functions of other genes.

Purpose To examine the role of tumor necrosis factor alpha (TNFalpha) in stromal neovascularization in injured cornea in vivo and in cytokine-enhanced vessel-like endothelial cell tube formation in vitro. Methods An in vitro model of angiogenesis was used to examine the roles of TNFalpha on tube formation by human umbilical vein endothelial cells (HUVECs) cocultured with fibroblasts on induction by transforming growth factor beta1 (TGFbeta1) and vascular endothelial growth factor (VEGF). Central cauterization was used to induce stromal neovascularization in corneas of wild-type (WT) and TNFalpha-null (Tnfalpha(-/-)) mice. At 7, 14, or 21 days of injury, experimental mice were killed, and the eyes were enucleated and subjected to histologic and immunohistochemical examination and real-time reverse transcription-polymerase chain reaction. Results HUVECs formed a vessel-like tube structure on the fibroblast feeder layer. Adding TGFbeta1, VEGF, or both augmented vessel-like tube formation by HUVECs cocultured with fibroblasts. Adding TNFalpha (5 ng/mL) completely abolished the formation of tube-like structures despite the presence or absence of TGFbeta1 or VEGF in coculture. In vivo, cauterization of the central cornea induced the formation of CD31(+) new vessels surrounding the limbus in WT mice. More prominent central stromal neovascularization accompanied by increased expression of TGFbeta1 and VEGF was found in Tnfalpha(-/-) mice compared with WT mice. Conclusions In addition to inhibiting TGFbeta1 and VEGF expression by fibroblasts, endogenous TNFalpha may counter the induction effects of TGFbeta1 and VEGF on vascular endothelial cells and may block neovascularization.

We previously reported that osteopontin (OPN), a matrix structural glycophosphoprotein, is upregulated in the injured mouse lens prior to the epithelial-mesenchymal transition (EMT). Here, we investigated the role of this protein in EMT of the lens epithelium during wound healing. The crystalline lens was injured by needle puncture in OPN-null (KO, n=40) and wild-type (WT, n=40) mice. The animals were killed at day 1, 2, 5, and 10 postinjury. Immunohistochemistry was employed to detect alpha-smooth muscle action (alphaSMA), a marker of EMT, collagen type I, transforming growth factor beta1 (TGFbeta1), TGFbeta2, and phospho-Smad2/3. Cell proliferation was assayed by examining uptake of bromodeoxyuridine (BrdU). The results showed that injury-induced EMT of mouse lens epithelium, as evaluated by histology, expression pattern of alphaSMA and collagen I, was altered in the absence of OPN with reduced phospho-Smad2/3 signaling. Upregulation of TGFbeta1 and TGFbeta2 in the epithelium was also inhibited. Cell proliferation was more active in KO mice as compared with WT mice at day 1 and 2, but not at day 5 and 10. An in vitro experiment shows OPN facilitates cell adhesion of lens epithelial cell line. OPN is required for activation of Smad2/3 signal in an injured lens epithelium and lens cell EMT.

PURPOSE. To investigate the effects of loss of osteopontin (OPN) in the healing of the injured cornea in mice. Cell culture study was also conducted to clarify the effects of OPN in fibroblast behaviors. METHODS. Ocular fibroblasts from wild-type (WT) and OPN-null (KO) mice were used to study the role of OPN on cell behavior. The effect of the lack of OPN on corneal wound healing was evaluated in mice. RESULTS. In cell culture, OPN is involved in cell adhesion and in the migration of ocular fibroblasts. Adhesion of the corneal epithelial cell line was not affected by exogenous OPN. OPN was upregulated in a healing, injured mouse cornea. Loss of OPN did not affect epithelial healing after simple epithelial debridement. Healing of an incision injury in cornea was delayed, with less appearance of myofibroblasts and transforming growth factor 1 expression in a KO mouse than in a WT mouse. The absence of OPN promoted tissue destruction after an alkali burn, resulting in a higher incidence of corneal perforation in KO mice than in WT mice. CONCLUSIONS. OPN modulates wound healing-related fibroblast behavior and is required to restore the physiological structure of the cornea after wound healing. (Invest Ophthalmol Vis Sci. 2008;49:1367‐1375) DOI:10.1167/iovs.07-1007

A series of biochemical analyses were carried out with keratoconus and normal corneas to determine the amount of stromal collagen, degree of posttranslational modification of collagen and the solubility of collagen. Our results revealed there was no obvious alteration in the degree of posttranslational modification of collagen in keratoconus corneas. However, the amount of collagen decreases and solubility of collagen increases in keratoconus corneas. It was also found that keratoconus corneas in organ culture produce substantially more collagenase and gelatinase activities than normal corneas. Our results suggest that keratoconus may represent a collagenolytic disease.

Escherichia coli were activated by incubation with 1% glutaraldehyde, and the activated bacteria were then conjugated by incubation with IgG. About 66,000 molecules of IgG were bound per cell. The Escherichia coli -antibody conjugates were then used in competitive radioimmune assays. When a specific IgG fraction was used to prepare the conjugate, each cell bound about 16,000 molecules of antigen. With an antigen which had a specific activity of about 300 cpm/ng (or about 4.8 × 10 4 cpm/pmol), the assay was linear over a range of 40–300 ng/ml (0.3–2.0 pmol/ml). About 100 samples were assayed per day.

Fibroblasts isolated by enzymic digestion of chick embryo tendons were incubated for several hours in suspension under conditions in which they were in steady state in terms of the synthesis and secretion of procollagen. Under these conditions, the cells synthesized and secreted about 630 microgram of procollagen/10(9) cells/h. The cells were labeled with [14C]proline for 15 to 120 min and then the kinetics of secretion were followed by chasing the label and assaying the 14C-peptides digestible by collagenase in the cells and in the medium. The results demonstrated that secretion of collagenase-digestible peptides did not follow the kinetics of a single first order process but suggested at least two pseudo-first order process with half-times of 14 and 115 min. The [14C]procollagen secreted during 0 to 30 min and 90 to 120 min of chase was the same in terms of the ratio of pro-alpha1 to pro-alpha2 chains, the size of the pro-alpha chains, the extent of interchain disulfide bonding, the extent of prolyl hydroxylation, and the degree of helicity as tested by resistance to pepsin digestion. Addition of ascorbic acid to the incubation medium increased slightly the extent of prolyl hydroxylation but did not alter the kinetics of secretion. The results suggested that the kinetics of secretion are influenced by a two-compartment system in which at least one metabolic pool contributing to the secretory process is present as a "side pocket."

An improved procedure was used to assay prolyl hydroxylase activity in both early-log and late-log L-929 fibroblasts grown on plastic surfaces. When 40 μg/ml of ascorbate was added to early-log phase cultures, the rate of hydroxy-[ 14 C] proline synthesis increased 2-fold within 4 h, but there was no change in prolyl hydroxylase activity per cell. The results indicated therefore that ascorbate did not “activate” prolyl hydroxylase in the sense of converting inactive enzyme protein to active enzyme protein. Instead ascorbate appeared to increase hydroxyproline synthesis in early-log L-929 fibroblasts because the prolyl hydroxylase reaction in such cells was limited by the availability of ascorbate or a similar cofactor. When 40 μg/ml of ascorbate was added to late-log phase cultures, there was essentially no effect on the rate of hydroxyl[ 14 C]-proline synthesis or prolyl hydroxylase activity. The late-log phase cells, however, contained three times more enzyme activity and about two times more immuno-reactive enzyme protein than early-log phase cells. In addition, the rate of protein synthesis per cell in late-log phase cells was only one-tenth the rate in early-log phase cells. The results suggested that as the cells grew to confluency, collagen polypeptides were more completely hydroxylated in part because the rate of polypeptide synthesis decreased and at the same time prolyl hydroxylase activity per cell increased. The results appear to provide an alternate explanation for previous observations on the effects of ascorbate and “crowding” on hydroxy[roline synthesis in cultures of L-929 fibroblasts.

Embryonic-chick tendon cells were incubated in suspension for 4h with (14)C-labelled amino acids, cell extracts were subjected to gel filtration, and the effluent was examined by rocket immunoelectrophoresis by using antibodies specific for the beta-subunit of chick prolyl hydroxylase. Two peaks of immunoreactive protein were found. The first peak contained 40% of the immunoreactive protein eluted from the column and 100% of the enzyme activity. Polyacrylamide-slab-gel electrophoresis in sodium dodecyl sulphate of an immunoprecipitate of this peak demonstrated that it consisted of the tetrameric form of prolyl hydroxylase, subunit composition alpha(2)beta(2) where alpha and beta are non-identical subunits. Only the alpha-subunits were labelled, indicating that they were synthesized during the 4h labelling period. The beta-subunits were unlabelled, indicating that they had been synthesized before the labelling period. The second peak eluted from the gel-filtration column contained 60% of the immunoreactive protein eluted from the column and was enzymically inactive. Polyacrylamide-slab-gel electrophoresis of an immunoprecipitate of this peak indicated that it consisted of a single labelled polypeptide chain, identified as cross-reacting protein, which was related to, but not identical with, the beta-subunit of prolyl hydroxylase. Pulse-chase experiments were performed on cultured chick tendon cells to demonstrate that alpha-subunits and cross-reacting protein had half-lives of about 60h. The half-life of beta-subunits was considerably longer, and the kinetic pattern was consistent with their being derived from a labelled precursor such as cross-reacting protein. The data presented here indicate that the active tetrameric form of prolyl hydroxylase in cells is assembled from alpha-subunits which are newly synthesized, and from beta-subunits which are derived from cross-reacting protein.

An improved procedure was developed to extract prolyl hydroxylase from tendon cells of chick embryos with detergent, and improved assays were developed for both the activity of the enzyme and the amount of enzyme protein. Freshly isolated tendon cells were found to contain approx. 100 μg of enzyme protein per 108 cells and 40–50% of the enzyme protein was active. When the cells were cultured, they were found to contain the same amount of enzyme protein by only 15–20% of the enzyme protein was active. Gel filtration of cell extracts indicated that the active form of prolyl hydroxylase in freshly isolated tendon cells and in cultured tendon cells had the same apparent size and the same activity per μg of immunoreactive protein as enzyme which was shown to be a tetramer. The inactive form was found to have about the same apparent size as subunits of the enzyme. When freshly isolated cells were incubated for 2 h in the presence of 40 μg per ml of ascorbate, there was a slight increase in the rate of hydroxyproline synthesis. In cultured cells, ascorbate at a concentration of 40 μg per ml caused a 2-fold increase in the rate of hydroxyproline synthesis within 30 min. However, ascorbate did not increase the activity of prolyl hydroxylase in extracts from either cell system. Therefore it appears that the influence of ascorbate on synthesis of procollagen hydroxyproline by the cells studied here must be ascribed to a cofactor effect on the hydroxylation reaction similar to that observed with purified enzyme, and it does not involve “activation” of inactive enzyme protein to active enzyme as has been observed in cultures of L-929 and 3T6 mouse fibroblasts.

Incorporation of a proline analog into collagen polypeptides was studied by incubating matrix-free tendon cells from 17-day-old chick embryos with cis-4-hydroxy- l -proline. Velocity sedimentation of intracellular polypeptides provided further evidence that incorporation of the analog into protein prevented the pro-α- and pro-γ-chains of procollagen from folding into a stable triple-helical conformation. The size of the newly synthesized intracellular and extracellular protein was examined under conditions which prevented proteolysis during processing of the samples. In contrast to previous observations, the results demonstrated that there was little if any intracellular degradation of nonhelical pro-α-and pro-γ-chains containing the proline analog, and a fraction of the nonhelical pro-γ-chains was secreted into the medium without extensive degradation. In further studies, the cells were incubated with 14C lysine, and the synthesis of glycosylated hydroxylysyl residues was measured in control cells and in cells incubated with cis-4-hydroxy- l -proline. The results demonstrated that the content of glycosylated hydroxylysyl residues in nonhelical pro-γ-chains containing cis-4-hydroxy- l -proline was increased twofold as compared to the triple-helical procollagen in control cells. The results suggested that under control conditions folding into the triplehelical conformation limits the extent of glycosylation of collagen. If folding is prevented or delayed, procollagen polypeptides are more extensively glycosylated.

Tissue morphogenesis during development is regulated by growth factors and cytokines, and is characterized by constant remodeling of extracellular matrix (ECM) in response to signaling molecules, for example, growth factors, cytokines, and so forth. Proteoglycans that bind growth factors are potential regulators of tissue morphogenesis during embryonic development. In this study, we showed that transgenic mice overexpressing biglycan under the keratocan promoter exhibited exposure keratitis and premature eye opening from noninfectious eyelid ulceration due to perturbation of eyelid muscle formation and the failure of meibomian gland formation. In addition, in vitro analysis revealed that biglycan binds to TGF-alpha, thus interrupting EGFR signaling pathways essential for mesenchymal cell migration induced by eyelid epithelium. The defects of TGF-alpha signaling by excess biglycan were further augmented by the interruption of the autocrine or paracrine loop of the EGFR signaling pathway of HB-EGF expression elicited by TGF-alpha. These results are consistent with the notion that under physiological conditions, biglycan secreted by mesenchymal cells serves as a regulatory molecule for the formation of a TGF-alpha gradient serving as a morphogen of eyelid morphogenesis.

Prolyl hydroxylase activity in cultured L-929 cells was found to increase when cells grew from log phase to stationary phase and when cells were harvested at the mid-log phase and replated at higher cell densities. Cycloheximide and actinomycin D inhibited the cell density-dependent increase in prolyl hydroxylase activity indicating that the increase in prolyl hydroxylase activity required de novo synthesis of protein and RNA. Prolyl hydroxylase was purified from cultured L-929 cells and antibodies against the protein were raised in rabbits. The antibodies were used to demonstrate that L-929 cells contained two forms of prolyl hydroxylase: an enzymatically active, tetrameric form consisting of two alpha and two beta polypeptide chains and an enzymatically inactive form containing immunologically cross-reacting protein. The polypeptide chains alpha, beta and cross-reacting protein were obtained by immunoadsorption. Peptide map analysis indicated that cross-reacting protein was similar if not identical to beta in primary structure, and alpha was different from both beta and cross-reacting protein. The results suggested that the prolyl hydroxylase levels in cells or tissues may be regulated by new protein and/or RNA synthesis.

Corneas and tendons dissected from 17-day-old chick embryos were labeled with [35S]methionine in the presence of 0.3 mM alpha,alpha'-dipyridyl. The unhydroxylated, 35S-labeled pro alpha chains and alpha chains were isolated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The pro alpha and alpha chains were then subjected to peptide-map analysis by proteolytic digestion with trypsin and alpha-chymotrypsin, papain or proteinase K. The peptide-maps derived from cornea and tendon pro alpha 1(I) chains are identical. Similar results were obtained from cornea and tendon alpha 1(I) chains. There were differences in the peptide maps derived from cornea and tendon pro alpha 2(I) chains. However, no difference was observed in alpha 2(I) chains. These results suggest that cornea and tendon pro alpha 1(I) chains are probably identical in primary structures, whereas the cornea pro alpha 2(I) chain may be different from the tendon pro alpha 2(I) chain within pepsin sensitive regions of the procollagen molecule. The reason for difference in the peptide-maps of pro alpha 2(I) chains remain unknown. One of the possible explanations is the variation of posttranslational modification within the propeptides of the pro alpha 2(I) chain. However, this hypothesis needs to be further investigated. Nevertheless, the finding that the peptide-maps of alpha 2(I) chains from tendons and corneas are identical fail to support the two genes hypothesis for pro alpha 2(I) chains.