Immunohistological analysis of transglutaminase factor XIIIA expression in mouse embryonic growth plate.

Previously we demonstrated the expression of Factor XIIIA (FXIIIA), a coagulation transglutaminase, in avian embryonic growth plate. To explore whether FXIIIA is also expressed by chondrocytes of the mammalian cartilage anlagen of bones, we analyzed the mouse embryonic growth plate by immunostaining using anti-FXIIIA antibodies developed against human and chicken proteins. We revealed the expression of FXIIIA in the epiphyseal growth plate, where FXIIIA appears first intracellularly in the zone of proliferation/maturation, and remains intra- and extracellularly throughout the hypertrophic zone. Externalization of FXIIIA occurs before mineralization. Transglutaminase activity was assayed in organ cultures using rhodamine-labeled synthetic substrate Pro-Val-Lys-Gly. Enzymatic activity shows a restricted distribution in cartilage and correlates with FXIIIA expression pattern, suggesting that cartilagenous transglutaminase activity is due, at least partially, to the FXIIIA isoform. We conclude, that coagulation factor FXIIIA is expressed by chondrocytes of embryonic mouse long bone cartilages in a strictly regulated pattern, which correlates with chondrocyte differentiation and matrix mineralization.

Transglutaminase factor XIIIA in the cartilage of developing avian long bones.

Previously, we showed that mRNA for transglutaminase factor XIIIA (FXIIIA) is up-regulated in the hypertrophic zone of the growth plate of the chicken tibiotarsus, a well-characterized model of long bone development. In the present study, we have studied the distribution of the FXIIIA protein and of transglutaminase enzymatic activity in this growth plate, as well as in the cartilage of the epiphysis, which includes that of the articular surface. By immunohistochemical analysis, the protein is detected in the zone of maturation, where it is mostly intracellular, and in the hypertrophic zone, where it is present both intracellularly and in the extracellular matrix. The intracellular enzyme is mostly a zymogen, as determined with an antibody specific for the activation peptide. Externalization of FXIIIA is accompanied by enzyme activation. To study the pattern of transglutaminase activity, a synthetic transglutaminase substrate, rhodamine-conjugated tetrapeptide (Pro-Val-Lys-Gly), was used for pulse labeling in organ cultures. Intensive incorporation of the fluorescent substrate was observed throughout the hypertrophic zone and in the cells surrounding the forming blood vessels. The patterns of FXIIIA immunostaining and substrate incorporation overlap almost completely. The cartilaginous factor XIIIA is different from the plasma form in that, both intracellularly and extracellularly, it exists as a monomer, as determined by Western analysis, whereas the plasma form of FXIII is a tetrameric complex composed of both A and B subunits. We also identified FXIIIA and transglutaminase activity within the articular and condylar regions of the tarsus, suggesting a possible involvement of mechanical pressure and/or stress in the production of the molecule and subsequent cross-linking of the cartilage matrix. Thus, transglutaminases, in particular FXIIIA, are involved in the formation of long bones through its activity both in the hypertrophic region of the growth plate and in the formation of articular/epiphyseal cartilages.

UDP-glucose pyrophosphorylase: up-regulation in hypertrophic cartilage and role in hyaluronan synthesis.

Previously, we have performed subtractive hybridization to identify genes up-regulated in hypertrophic chondrocytes of the avian epiphyseal growth plate. In the present study, we report the identification of one of the clones as UDP-glucose pyrophosphorylase (UDPG-PPase) and propose a possible function for this enzyme in regulating hyaluronan (HA) synthesis in hypertrophic cartilage. We have cloned the 2.6 kb full-length cDNA for avian UDPG-PPase and confirmed its up-regulation in hypertrophic versus non-hypertrophic cartilage by Northern-blot analysis. The 6-fold increase in mRNA was paralleled by an equivalent increase in enzymic activity. The enzyme catalyses the conversion of glucose 1-phosphate into UDP-glucose, which is used to synthesize a number of cellular components, including HA. Overexpression of enzymically active UDPG-PPase in non-hypertrophic chondrocytes resulted in a 2-3-fold increase in total HA, as determined by a competitive binding assay and immunohistochemistry. In the developing growth plate, HA synthesis was elevated in the hypertrophic zone along with the up-regulation of the HA synthase (HAS)-2 gene. Our data suggest that an increase in both activities, UDPG-PPase and HAS-2, is required for non-hypertrophic chondrocytes to synthesize an amount of HA comparable with that in hypertrophic chondrocytes. Therefore we conclude that HA synthesis during chondrocyte differentiation is regulated at the level of the substrate-provider gene, UDPG-PPase, as well as the HAS genes.

Nuclear translocation of ferritin in corneal epithelial cells.

Our previous studies have shown that ferritin within developing avian corneal epithelial cells is predominantly a nuclear protein and that one function of the molecule in this location is to protect DNA from UV damage. To elucidate the mechanism for this tissue-specific nuclear translocation, cultured corneal epithelial cells and corneal fibroblasts were transfected with a series of deletion constructs for the heavy chain of ferritin, ferritin-H, tagged with a human c-myc epitope. The subcellular localization of the ferritin was determined by immunofluorescence for the myc-tag. For the corneal epithelial cells, the first 10 or the last 30 amino acids of ferritin-H could be deleted without affecting the nuclear localization. However, larger deletions of these areas, or deletions along the length of the body of the molecule, resulted largely in retention of the truncated proteins within the cytoplasm. Thus, it seems that no specific region functions as an NLS. Immunoblotting analysis of SDS-PAGE-separated extracts suggests that assembly of the supramolecular form of ferritin is not necessary for successful nuclear translocation, because one deletion construct that failed to undergo supramolecular assembly showed nuclear localization. In transfected fibroblasts, the endogenous ferritin remained predominantly in the cytoplasm, as did that synthesized from transfected full-length ferritin constructs and from two deletion constructs encoding truncated chains that could still assemble into the supramolecular form of ferritin. However, those truncated chains that were unable to participate in supramolecular assembly generally showed both nuclear and cytoplasmic localization, indicating that, in this cell type, supramolecular assembly is involved in restricting ferritin to the cytoplasm. These data suggest that for corneal epithelial cells, the nuclear localization of ferritin most likely involves a tissue-specific mechanism that facilitates transport into the nucleus, whereas, in fibroblasts, the cytoplasmic retention involves supramolecular assembly that prevents passive diffusion into the nucleus.

Regulation of endochondral cartilage growth in the developing avian limb: cooperative involvement of perichondrium and periosteum.

The perichondrium and periosteum have recently been suggested to be involved in the regulation of limb growth, serving as potential sources of signaling molecules that are involved in chondrocyte proliferation, maturation, and hypertrophy. Previously, we observed that removal of the perichondrium and periosteum from tibiotarsi in organ culture resulted in an overall increase in longitudinal cartilage growth, suggesting negative regulation originating from these tissues. To determine if the perichondrium and periosteum regulate growth through the production of diffusible factors, we have tested various conditioned media from these tissues for the ability to modify cartilage growth in tibiotarsal organ cultures from which these tissues have been removed. Both negative and positive regulatory activities were detected. Negative regulation was observed with conditioned medium from (1) cell cultures of the region bordering both the perichondrium and the periosteum, (2) co-cultures of perichondrial and periosteal cells, and (3) a mixture of conditioned media from perichondrial cell cultures and periosteal cell cultures. The requirement for regulatory factors from both the perichondrium and periosteum suggests a novel mechanism of regulation. Positive regulation was observed with conditioned media from several cell types, with the most potent activity being from articular perichondrial cells and hypertrophic chondrocytes.

Complete primary structure of the chicken alpha1(V) collagen chain.

Chicken alpha1(V) collagen cDNAs have been cloned by a variety of methods and positively identified. We present here the entire translated sequence of the chick polypeptide and compare selected regions to other collagen chains in the type V/XI family.

Distribution of type IV collagen during avian limb bud development.

Normal limb development is dependent on an epithelial-mesenchymal interaction between the overlying apical ectodermal ridge (AER) and the underlying mesenchyme. The basement membrane between the epithelium and the mesenchyme has been proposed to play an important role in regulating epithelial-mesenchymal interactions during development. To explore the role basement membrane type IV collagen may play during limb development we investigated the distribution of type IV collagen by immunolocalization. Developing avian leg buds were examined at 2 developmental stages: stage 23, when the AER is inductively active, and stage 28, when the AER is regressing. The proximal basement membrane in stage 23 limb buds stained much more intensely than the distal basement membrane. This proximal-distal immunostaining difference was less in stage 28 limb buds. We used the monoclonal antibody IIB12, which recognizes an epitope adjacent to the initial collagenase cleavage site on the type IV collagen molecule, to explore whether this proximal-distal difference in basement membrane staining could result from the loss of type IV collagen. The distal basement membrane of stage 23 limb buds demonstrated little immunostaining with the IIB12 antibody, suggesting enhanced collagenase-associated degradation. The immunostaining was increased in stage 28 limb buds. Consistent with a loss of type IV collagen, we also found that unfixed stage 23 leg bud cryostat sections stored at 4 degrees C lost their immunostaining for type IV collagen, in contrast to stored stage 28 limb bud cryostat sections. These results demonstrate that type IV collagen is distributed in a proximal-distal pattern in the basement membrane of the developing chick limb bud and suggest that this pattern may be the result of a selective degradation of type IV collagen in the basement membrane underlying the active AER. These results are consistent with the hypothesis that the basement membrane plays a role in regulating the epithelial-mesenchymal interaction responsible for induction of limb outgrowth.

Development and roles of collagenous matrices in the embryonic avian cornea.

Corneal development requires the production, assembly and sometimes replacement of a number of collagenous matrices. The embryonic chick cornea is well-characterized and offers certain advantages for studying the assembly and roles of these matrices. We will first describe the matrices to be examined. These include the corneal stroma proper, first formed as the primary stroma and subsequently as the secondary (mature) stroma; Bowman's Membrane; Descemet's Membrane; and the hemidesmosome of the epithelial cell attachment complex. We will then describe the characteristics of the collagen types involved, including: the fibrillar collagens (types I, II and V), the fibril-associated collagens (types IX, XII and XIV), and the transmembrane collagen of the hemidesmosome (type XVII). Then, in each subsequent section we will examine in detail the structure, assembly and development of each collagenous matrix, and how each specific collagen and/or combination of collagens are thought to provide the matrices with their unique properties. The work and views presented here are largely from our own laboratories. Thus, this article is not meant to be a comprehensive review of the literature. For pertinent references by others, when possible, we will cite recent reviews.

Plasma transglutaminase in hypertrophic chondrocytes: expression and cell-specific intracellular activation produce cell death and externalization.

We previously used subtractive hybridization to isolate cDNAs for genes upregulated in chick hypertrophic chondrocytes (Nurminskaya, M. , and T.F. Linsenmayer. 1996. Dev. Dyn. 206:260-271). Certain of these showed homology with the "A" subunit of human plasma transglutaminase (factor XIIIA), a member of a family of enzymes that cross-link a variety of intracellular and matrix molecules. We now have isolated a full-length cDNA for this molecule, and confirmed that it is avian factor XIIIA. Northern and enzymatic analyses confirm that the molecule is upregulated in hypertrophic chondrocytes (as much as eightfold). The enzymatic analyses also show that appreciable transglutaminase activity in the hypertrophic zone becomes externalized into the extracellular matrix. This externalization most likely is effected by cell death and subsequent lysis-effected by the transglutaminase itself. When hypertrophic chondrocytes are transfected with a cDNA construct encoding the zymogen of factor XIIIA, the cells convert the translated protein to a lower molecular weight form, and they initiate cell death, become permeable to macromolecules and eventually undergo lysis. Non-hypertrophic cells transfected with the same construct do not show these degenerative changes. These results suggest that hypertrophic chondrocytes have a novel, tissue-specific cascade of mechanisms that upregulate the synthesis of plasma transglutaminase and activate its zymogen. This produces autocatalytic cell death, externalization of the enzyme, and presumably cross-linking of components within the hypertrophic matrix. These changes may in turn regulate the removal and/or calcification of this hypertrophic matrix, which are its ultimate fates.

Regulation of growth region cartilage proliferation and differentiation by perichondrium.

Endochondral bone formation in vertebrates requires precise coordination between proliferation and differentiation of the participating chondrocytes. We examined the role of perichondrium in this process using an organ culture system of chicken embryonic tibiotarsi. A monoclonal antibody against chicken collagen type X, specifically expressed by hypertrophic chondrocytes, was utilized to monitor the terminal differentiation of chondrocytes. Proliferation of chondrocytes was examined by a BrdU-labeling procedure. The absence of perichondrium is correlated with an extended zone of cartilage expressing collagen type X, suggesting that the perichondrium regulates chondrocyte hypertrophy in a negative manner. Removal of perichondrium, in addition, resulted in an extended zone of chondrocytes incorporating BrdU, indicating that the perichondrium also negatively regulates the proliferation of chondrocytes. Partial removal of perichondrium from one side of the tibiotarsus led to expansion of both the collagen type X-positive domain and the BrdU-positive zone at the site of removal but not where the perichondrium remained intact. This suggests that both types of regulation by the perichondrium are local effects. Furthermore, addition of bovine parathyroid hormone (PTH) to perichondrium-free cultures reversed the expansion of the collagen type X-positive domain but not that of the proliferative zone. This suggests that the regulation of differentiation is dependent upon the PTH/PTHrP receptor and that the regulation of proliferation is likely independent of it. Taken together, these results are consistent with a model where perichondrium regulates both the exit of chondrocytes from the cell cycle, and their subsequent differentiation.

Collagen type IX and developmentally regulated swelling of the avian primary corneal stroma.

A critical event in avian corneal development occurs when the acellular primary stroma swells and becomes populated by mesenchymal cells that migrate from the periphery. These cells then deposit the mature stromal matrix that exhibits the unique features necessary for corneal function. Our previous work correlated the disappearance of collagen type IX immunoreactivity at stage 27 (5 1/2-6 days) with matrix swelling and invasion. To investigate further the mechanism of this disappearance, we employed immunohistochemistry after tissue fixation with Histochoice, a non-crosslinking fixative, immunoblot analysis of protein extracts, and gel substrate chromatography (zymography) to detect endogenous proteolytic activity. We found that corneas fixed in Histochoice retain immunoreactivity for type IX collagen for 1-2 days after corneal swelling. This immunoreactivity, however, becomes extractable from tissue sections of unfixed corneas at the time of initiation of stromal swelling and mesenchymal cell invasion. Immunoblot analysis confirmed that, following swelling, immunoreactivity for collagen IX decreased substantially in corneas, but not in the vitreous body, which served as a comparison. Analysis of ammonium sulfate (AS) fractions of such extracts indicated that, at the time of swelling, much of the immunoreactivity for type IX collagen in cornea shifted from the AS precipitate (containing high molecular weight molecules) to the AS supernatant (containing smaller fragments). In contrast, collagen IX immunoreactivity from the vitreous was precipitated by ammonium sulfate throughout the period of study. Collagen type II, a major fibrillar collagen in both the corneal stroma and vitreous, remained in the high molecular weight fraction at all times examined. Zymography detected the presence of the latent (proenzyme) form of gelatinase A (MMP-2) before corneal swelling and invasion (4 days), and both the latent and active forms of the enzyme after corneal swelling. This suggests tissue-specific, developmentally regulated proteolysis of collagen IX as a trigger for corneal matrix swelling.

Type X collagen and other up-regulated components of the avian hypertrophic cartilage program.

Elucidating the cellular and molecular processes involved in growth and remodeling of skeletal elements is important for our understanding of congenital limb deformities. These processes can be advantageously studied in the epiphyseal growth zone, the region in which all of the increase in length of a developing long bone is achieved. Here, young chondrocytes divide, mature, become hypertrophic, and ultimately are removed. During cartilage hypertrophy, a number of changes occur, including the acquisition of synthesis of new components, the most studied being type X collagen. In this review, which is based largely on our own work, we will first examine the structure and properties of the type X collagen molecule. We then will describe the supramolecular forms into which the molecule becomes assembled within tissues, and how this changes its physical properties, such as thermal stability. Certain of these studies involve a novel, immunohistochemical approach that utilizes an antitype X collagen monoclonal antibody that detects the native conformation of the molecule. We describe the developmental acquisition of the molecule, and its transcriptional regulation as deduced by in vivo footprinting, transient transfection, and gel-shift assays. We provide evidence that the molecule has unique diffusion and regulatory properties that combine to alter the hypertrophic cartilage matrix. These conclusions are derived from an in vitro system in which exogenously added type X collagen moves rapidly through the cartilage matrix and subsequently produces certain changes mimicking ones that have been shown normally to occur in vivo. These include altering the cartilage collagen fibrils and effecting changes in proteoglycans. Last, we describe the subtractive hybridization, isolation, and characterization of other genes up-regulated during cartilage hypertrophy, with specific emphasis on one of these--transglutaminase.

Nuclear ferritin protects DNA from UV damage in corneal epithelial cells.

Previously, we identified the heavy chain of ferritin as a developmentally regulated nuclear protein of embryonic chicken corneal epithelial cells. The nuclear ferritin is assembled into a supramolecular form indistinguishable from the cytoplasmic form of ferritin found in other cell types and thus most likely has iron-sequestering capabilities. Free iron, via the Fenton reaction, is known to exacerbate UV-induced and other oxidative damage to cellular components, including DNA. Since corneal epithelial cells are constantly exposed to UV light, we hypothesized that the nuclear ferritin might protect the DNA of these cells from free radical damage. To test this possibility, primary cultures of cells from corneal epithelium and stroma, and from skin epithelium and stroma, were UV irradiated, and DNA strand breaks were detected by an in situ 3'-end labeling method. Corneal epithelial cells without nuclear ferritin were also examined. We observed that the corneal epithelial cells with nuclear ferritin had significantly less DNA breakage than other cell types examined. Furthermore, increasing the iron concentration of the culture medium exacerbated the generation of UV-induced DNA strand breaks in corneal and skin fibroblasts, but not in the corneal epithelial cells. Most convincingly, corneal epithelial cells in which the expression of nuclear ferritin was inhibited became much more susceptible to UV-induced DNA damage. Therefore, it seems that corneal epithelial cells have evolved a novel, nuclear ferritin-based mechanism for protecting their DNA against UV damage.

Multiple transcriptional elements in the avian type X collagen gene. Identification of Sp1 family proteins as regulators for high level expression in hypertrophic chondrocytes.

During the cartilage-to-bone transition, participating chondrocytes eventually undergo hypertrophy and are replaced by bone and marrow. Type X collagen is synthesized by chondrocytes specifically when they become hypertrophic, and this specificity is primarily regulated at the level of transcription. Previously, we demonstrated that a proximal promoter region from nucleotide -562 to +86 contained cis-acting elements that directed high level expression of a reporter gene in a cell-specific manner (Long, F., and Linsenmayer, T. F. (1995) J. Biol. Chem. 270, 31310-31314). In the present study, we have further dissected this region by generating a series of constructs and examining their expression in hypertrophic versus nonhypertrophic chondrocytes. Several positive and negative elements have been delineated within the proximal promoter region to mediate the regulation of transcription in hypertrophic chondrocytes. Most notably, a sequence from nucleotide -139 to +5 was sufficient to direct high level expression in this cell type. Electrophoresis mobility shift assay and supershift experiments identified within this sequence two 10-base pair noncanonical binding sites for Sp1 proteins. Mutations within the Sp1 binding sites either diminished or abolished the expression driven by the sequence from -139 to +5. These results indicate that the Sp1 proteins mediate the cell-specific expression of type X collagen.

Ferritin is a developmentally regulated nuclear protein of avian corneal epithelial cells.

Previously, we generated monoclonal antibodies against chicken corneal cells (Zak, N. B., and Linsenmayer, T. F. (1983) Dev. Biol. 99, 373). We have now observed that one group of these antibodies reacts with a developmentally regulated component of corneal epithelial cell nuclei. This component is the heavy chain of ferritin, as determined by analyses of immunoisolated cDNA clones and immunoblotting of the protein. Immunoblotting also suggests that the nuclear ferritin may be in a supramolecular form that is similar to the iron-binding ferritin complex found in the cytoplasm of many cells. In vitro cultures and transfection studies show that the nuclear localization depends predominantly on cell type but can be altered by the in vitro environment. The appearance of nuclear ferritin is at least partially under translational regulation, as is known to be true for the cytoplasmic form of the molecule. The tissue and developmental distributions of the mRNA for the molecule are much more extensive than the protein itself, and the removal of iron from cultures of corneal epithelial cells with the iron chelator deferoxamine prevents the appearance of nuclear ferritin. At present the functional role(s) of nuclear ferritin remain unknown, but previous studies on cytoplasmic ferritin raise the possibility that it prevents damage due to free radical generation ("oxidative stress") by sequestering iron. Although it remains to be tested whether nuclear ferritin prevents oxidative damage, we find this an attractive possibility. Since the corneal epithelium is transparent and is constantly exposed to free radical-generating UV light, it is possible that the cells of this tissue have evolved a specialized mechanism to prevent oxidative damage to their nuclear components.

Type XVII collagen (BP 180) in the developing avian cornea.

PURPOSE: Previous sequence analyses of hemidesmosomal BP 180/collagen XVII cDNA from human skin and of a similar chicken corneal cDNA showed some similarities, but major differences as well. The authors examined whether, in one species, the same mRNA is present in cornea and skin. They also studied the developmental expression of the molecule and compared it to the transmembrane hemidesmosome component, alpha 6 beta 4 integrin, and to the formation of hemidesmosomes themselves. METHODS: Cornea and skin BP 180/collagen XVII cDNAs were cloned by reverse transcription-polymerase chain reaction (RT-PCR) and sequenced. Developmental expression was evaluated by quantitative RT-PCR, immunoblotting, and immunofluorescence microscopy. alpha 6 beta 4 integrin was evaluated by immunofluorescence microscopy, and hemidesmosome formation was assessed by electron microscopy. RESULTS: The same alpha 1 (XVII) collagen/BP 180 mRNA is present in cornea and skin. The appearance of alpha 1 (XVII) collagen mRNA and protein shows similar temporal patterns of expression, with changes in the mRNA preceding those of the protein by approximately 2 days. The appearance of mature hemidesmosomes lags still further. Immunofluorescence histochemistry of alpha 1 (XVII) collagen and alpha 6 beta 4 integrin shows that their developmental appearance is regulated closely. CONCLUSIONS: The differences between human BP 180/collagen XVII and the chicken corneal molecule represent species divergence. The appearance of alpha 1 (XVII) collagen mRNA and protein is regulated closely, with the protein lagging. Mature hemidesmosomes, once present, have a low turnover rate. The developmental appearance of alpha 1 (XVII) collagen and alpha 6 beta 4 integrin are regulated closely. However, the component responsible for initiating hemidesmosome formation remains unknown.

Reduction of type V collagen using a dominant-negative strategy alters the regulation of fibrillogenesis and results in the loss of corneal-specific fibril morphology.

A number of factors have been implicated in the regulation of tissue-specific collagen fibril diameter. Previous data suggest that assembly of heterotypic fibrils composed of two different fibrillar collagens represents a general mechanism regulating fibril diameter. Specifically, we hypothesize that type V collagen is required for the assembly of the small diameter fibrils observed in the cornea. To test this, we used a dominant-negative retroviral strategy to decrease the levels of type V collagen secreted by chicken corneal fibroblasts. The chicken alpha 1(V) collagen gene was cloned, and retroviral vectors that expressed a polycistronic mRNA encoding a truncated alpha 1(V) minigene and the reporter gene LacZ were constructed. The efficiency of viral infection was 30-40%, as determined by assaying beta-galactosidase activity. To assess the expression from the recombinant provirus, Northern analysis was performed and indicated that infected fibroblasts expressed high steady-state levels of retroviral mRNA. Infected cells synthesized the truncated alpha 1(V) protein, and this was detectable only intracellularly, in a distribution that colocalized with lysosomes. To assess endogenous alpha 1(V) protein levels, infected cell cultures were assayed, and these consistently demonstrated reductions relative to control virus-infected or uninfected cultures. Analyses of corneal fibril morphology demonstrated that the reduction in type V collagen resulted in the assembly of large-diameter fibrils with a broad size distribution, characteristics similar to fibrils produced in connective tissues with low type V concentrations. Immunoelectron microscopy demonstrated the amino-terminal domain of type V collagen was associated with the small-diameter fibrils, but not the large fibrils. These data indicate that type V collagen levels regulate corneal fibril diameter and that the reduction of type V collagen is sufficient to alter fibril assembly so that abnormally large-diameter fibrils are deposited into the matrix.

Positive regulation of corneal type V collagen mRNA: analysis by chicken-human heterokaryon formation.

Our previous studies have suggested that type V collagen is at least one factor responsible for the characteristically small, uniform diameter of striated collagen fibrils of the corneal stroma. These fibrils, which are heterotypic combinations of collagen types I and V, contain four- to fivefold more type V collagen than those of tendon and sclera. The latter are much larger and more heterodisperse. This high content of type V collagen in cornea is reflected by an equally elevated content of alpha1(V) chain mRNA in corneal fibroblasts. Thus, the increased production of the molecule in cornea appears to be regulated at the level of transcription and/or mRNA stability. One possible explanation for this is that corneal fibroblasts contain positive regulatory factors that specifically upregulate transcription of the type V collagen genes and/or increase their mRNA stability. To test this possibility, we have produced transient heterokaryons by fusing chicken corneal fibroblasts with two human noncorneal cell lines selected as containing little if any alpha1(V) mRNA. If the chicken corneal cells contain positive regulators that can act across species, these regulators should result in increased levels of the human alpha1(V) transcript. The results were evaluated by reverse transcript-polymerase chain reaction employing a primer pair selected for its ability specifically to amplify part of the human alpha1(V) mRNA. In fusions between chicken corneal fibroblasts and the human cell lines, after a lag of 10-14 h the heterokaryon-containing cultures showed de novo appearance or upregulation of human alpha1(V) chain mRNA, compared with that of the parental cell lines. Cultures of the mixed cell types that had not been fused showed no such upregulation, so the effect was not mediated by diffusible substances acting between the cells. Chicken tendon fibroblasts, a low producer of type V collagen, when tested in the same assay, evoked no detectible increase in the human transcript. Thus, corneal cells do contain positive regulators for alpha1(V) chain mRNA, and this effect is at least somewhat cell specific.

Characterization and developmental regulation of avian corneal beta-1,4-galactosyltransferase mRNA.

Corneal development involves the synthesis and assembly of a number of specialized extracellular matrices. These matrices have distinctive properties derived from a unique assembly of collagens, proteoglycans and glycoproteins. The synthesis of each of these requires a number of enzymes. By probing a corneal cDNA library for genes that appeared to be up-regulated in cornea we have isolated a cDNA that represents an mRNA encoding the enzyme beta-1,4-galactosyltransferase. In cornea, a major function for this enzyme is likely to be in the synthesis of the keratan sulfate proteoglycan, lumican. Employing quantitative reverse transcript-polymerase chain reaction, we have observed that the steady-state level of mRNA for the molecule is elevated during certain stages of corneal development. It is also elevated in corneal fibroblasts in culture that have a greatly decreased synthesis of the mature lumican molecule. These data are consistent with, and complement, studies by others that show a corresponding regulation of the lumican core protein during development and in corneal fibroblast cultures.

Identification and characterization of up-regulated genes during chondrocyte hypertrophy.

Chondrocyte hypertrophy involves de novo acquisition and/or increased expression of certain gene products including, among others, type X collagen, alkaline phosphatase, and matrix metalloproteinases. To analyze further the genetic program associated with chondrocyte hypertrophy, we have employed a modification of the polymerase chain reaction-mediated subtractive hybridization method of Wang and Brown (Wang and Brown [1991] Proc. Natl. Acad. Sci 88:11505). Cultures of hypertrophic tibial chondrocytes and nonhypertrophic sternal cells were used for poly A+ RNA isolation. Among 50 individual cDNA fragments isolated for up-regulated hypertrophic genes, 18 were tentatively identified by their similarities to entries in the GenBank database, whereas the other 32 showed no significant similarity. The identified genes included translational and transcriptional regulatory factors, ribosomal proteins, the enzymes transglutaminase and glycogen phosphorylase, type X collagen (highly specific for hypertrophic cartilage matrix), gelsolin, and the carbohydrate-binding protein galectin. Two of these, transglutaminase and galectin, were cloned and were further characterized. The chondrocyte transglutaminase revealed previously in hypertrophic cartilage by immunochemical methods appears to be the chicken equivalent of mammalian factor XIIIa (showing 75% overall protein similarity). The chicken chondrocyte galectin is a variant of mammalian galectin-3. Galectins are known to bind to components found in hypertrophic cartilage, and factor XIIIa is known to crosslink some of the same components, possibly modifying them for calcification and/or removal.