Role of p27(Kip1) in cAMP- and TGF-beta2-mediated antiproliferation in rabbit corneal endothelial cells.

PURPOSE: To determine whether p27(Kip1) plays a role in antiproliferation mediated by antimitogens (cAMP and TGF-beta2) in rabbit corneal endothelial cells (CECs). METHODS: Cell proliferation was assayed using a colorimetric method to determine the number of viable cells. Subcellular localization of cell cycle-regulatory proteins was determined by immunofluorescent staining, and expression of the proteins was analyzed by immunoblot analysis. RESULTS: When cells were treated with cAMP or TGF-beta2, serum-mediated cell proliferation was inhibited in a dose-dependent manner. Simultaneous treatment of the two antimitogens did not show a synergistic effect on inhibition of cell growth. Expression of cell cycle-regulatory proteins, such as cyclin-D1, cyclin-E, cdk2, cdk4, p21(Cip1), and p27(Kip1) was determined using immunofluorescent staining. A strong nuclear staining was observed for p27(Kip1). The other proteins were not stained or were only very faintly stained. Treatment of cells with either cAMP or TGF-beta2 did not change the staining potential of any proteins other than p27(Kip1), but all cells were positive for nuclear p27(Kip1) when treated with either TGF-beta2 or cAMP. In contrast, mitogen (FGF-2)-containing medium decreased the number of p27(Kip1)-positive cells. When the expression level of p27(Kip1) was determined using immunoblot analysis in the cells treated either with FGF-2 alone or with a concomitant treatment with FGF-2 and cAMP for 24 hours, FGF-2 markedly decreased the p27(Kip1) level, and cAMP prevented the decrease in p27(Kip1) level induced by FGF-2. No such phenomenon occurred during a short-term exposure of cells to either FGF-2 or cAMP or to a combination of the two. When cells were stained for phosphorylated p27(Kip1), FGF-2 markedly enhanced the staining of phosphorylated p27(Kip1) in nuclei, whereas both cAMP and TGF-beta2 prevented the phosphorylation of p27(Kip1). CONCLUSIONS: These findings suggest that both antimitogens upregulate the expression of p27(Kip1) as they prevent the decrease of the p27(Kip1) level mediated by mitogen. Furthermore, cAMP and TGF-beta2 may inhibit the G(1)-to-S transition by blocking phosphorylation of p27(Kip1), which is a prerequisite for nuclear export of the inhibitor molecule for degradation.

Epitheliomesenchymal transdifferentiation of cultured RPE cells.

Retinal pigment epithelium (RPE) cells of the proliferative vitreoretinopathy (PVR) membrane take on the shape of fibroblasts and participate in fibrosis, thus deviating from the character of epithelial cells. This study was undertaken to evaluate RPE cell transdifferentiation in vitro. During the culture of porcine RPE cells, primary and 10th-passaged RPE cells were investigated for cell growth in response to transforming growth factor (TGF) beta(2), change of phenotype and amount in collagen synthesis as well as expression of alpha-smooth-muscle actin (alpha-SMA). TGF-beta(2) inhibited the proliferation of the primary cultures of RPE cells in a dose-dependent manner, while the spindle-shaped 10th-passaged RPE cells were not inhibited by TGF-beta(2). The 10th-subcultured cells did not show much difference in the quality of collagen synthesis, other than type VIII collagen which was not produced. Collagen synthesis was dose-dependently stimulated by TGF-beta(2). The stimulation by TGF-beta(2) in the 10th-passaged RPE cells was much greater than in primary RPE cells. The 10th-subcultured RPE cells produced substantial alpha-SMA compared to alpha-SMA production by primary RPE cells. These results were also observed by confocal laser microscopy. These findings indicated that RPE metaplasia resulting in a change of biological cell behavior might be a necessary predisposing step in the development of PVR.

Craniofacial dysmorphogenesis in transgenic mice.

While using transgenic mice to study the regulation of alpha-fetoprotein (AFP) it was noted that two different alpha-fetoprotein-chloramphenicol acetyl transferase (CAT) transgenes resulted in the appearance of craniofacial anomalies in 11% of the offspring derived from crosses between transgenic mice and nontransgenic mates. A total of 13 fetuses exhibited abnormalities; two are described in detail. Ninety-two percent of the affected fetuses had some form of mandibular abnormality while zygomatic and ossicular defects appeared in more than 40% of the specimens. Aglossia and aberrant musculature were also present in the most severely involved specimen. Eight of the affected fetuses were screened for the presence of the AFP-CAT plasmid and all were found to be heterozygous for the transgene. Since the probability that all 8 of the abnormal fetuses known to carry the CAT gene would have done so by chance was only 1 in 256, it may be assumed that these anomalies did not appear spontaneously, but were somehow created by the transgenic procedure. It is not known how the transgenic material led to the observed dysmorphogenetic pattern, but theoretically introduction of the AFP-CAT plasmid could have disrupted morphogenesis through the presence of the "foreign" CAT protein or a decrease in the availability of AFP. Since AFP levels were found to be normal in both the liver and the yolk sac of transgenic fetuses, it appears that the presence of CAT was responsible for the craniofacial anomalies described here.

Common biochemical pathway of dysmorphogenesis in murine embryos: use of the glucocorticoid pathway by phenytoin.

Phenytoin (5,5-diphenylhydantoin), a common anticonvulsant drug, is known to produce anomalies in the craniofacial region of animals and humans. Furthermore, recent evidence suggests that phenytoin disrupts craniofacial and neural tube morphogenesis by inhibiting the arachidonic acid cascade, a pathogenesis already implicated for glucocorticoids and hyperglycemia in the palate. This study tested the hypothesis that phenytoin interferes with the arachidonic acid cascade via the same biochemical pathway demonstrated for glucocorticoids. The proposed pathway was tested at two levels. First, indomethacin, an inhibitor of the enzyme cyclooxygenase, was used in culture to block the correction of phenytoin-induced defects by arachidonic acid. Second, cortexolone, an anti-glucocorticoid that binds at the glucocorticoid receptor binding site, was tested for its ability to prevent phenytoin-induced teratogenicity. Eighty-four percent of the embryos cultured in phenytoin and 93% of those cultured in phenytoin plus arachidonic acid and indomethacin had neural tube and/or craniofacial deformities. In contrast, only 14% of the embryos cultured in phenytoin plus cortexolone were affected. Indomethacin itself produced anomalies in 83% of the exposed embryos. These data are consistent with the hypothesis that the teratogenic action of phenytoin in murine embryo cultures occurs via the glucocorticoid anti-inflammatory pathway. Thus, the glucocorticoid receptor appears to be responsible for mediating phenytoin-induced teratogenicity.

Dysmorphogenesis of the mandible, zygoma, and middle ear ossicles in hemifacial microsomia and mandibulofacial dysostosis.

A review of the anatomical changes in patients with various "first arch" syndromes shows that some anomalies (e.g., micrognathia, ear defects) generally appear together. This study tested the hypothesis that the mandible, zygomatic arch, and middle ear ossicles are a developmental field (i.e., when any of these structures is anomalous, the other two will be also). The hypothesis was tested using data from 25 patients with mandibulofacial dysostosis (MFD) and 40 patients with hemifacial microsomia (HFM). Analysis of the pooled data showed that the hypothesis of character association was generally supported. However, the medians suggested that different factors probably played a role in determining how these three anatomical structures were associated in MFD and HFM. Errors in chondrogenesis may have been primarily responsible for the HFM phenotype. Alterations in Meckel and palatoquadrate cartilages would account for the size and shape changes observed in the ossicles and mandible, while changes in cranial base cartilages may explain the changes noted in the zygomatic arch. Since all three structures were equally affected in MFD, it is problematic to use an interference with chondrogenesis as an explanation for the phenotype. We conclude that although the mandible, zygomatic arch, and middle ear ossicles appear to form a "developmental field," the association between structures varies for HFM and MFD. The relatively lesser involvement of the zygomatic arch in HFM than in MFD suggests different pathogeneses for the two diagnostic groups and maybe a useful criterion for judging animal models of HFM.

Arachidonic acid reversal of phenytoin-induced neural tube and craniofacial defects in vitro in mice.

Diphenylhydantoin (DPH), a common anticonvulsant drug, is known to produce anomalies in the craniofacial region of animals and humans. We hypothesize that phenytoin disrupts craniofacial morphogenesis by inhibiting the arachidonic acid cascade; a pathogenesis already demonstrated for glucocorticoids and hyperglycemia. This hypothesis was tested in vitro by administering DPH, with and without the addition of exogenous arachidonic acid (AA), to murine embryos. Forty-five 8.7-day-old embryos were randomly assigned to one of three groups: control, DPH, or DPH plus AA. After 48 hours in culture, all specimens were examined at 6x magnification for defects in the facial arches, head fold, and neural tube fusion. The DPH-treated specimens had a significantly greater (P less than or equal to .05) number of anomalies in each of the three anatomical areas than did the controls. Specimens cultured in DPH plus AA had significantly fewer defects in each of the three features than those treated with DPH alone. These data support the hypothesis that phenytoin disrupts normal embryonic development through interruption of the arachidonic acid cascade. Furthermore, removal of the visceral yolk sac from 50% of the specimens in control and treatment groups provided evidence that the drug had a direct effect on the embryos rather than secondary to yolk sac involvement as has been suggested in the literature.

Skeletal changes in the hindlimbs of bipedal rats.

Experimental bipedalism in rats has been proposed as a model for studying evolutionary and orthopedic change. This study quantified the skeletal changes which occur in the hindlimbs of these bipedal rats. Bipedalism was produced in 16 male rats by forelimb amputation during the neonatal period. The rats were sacrificed at maturity and the femurs and tibia-fibulas from randomly selected hindlimbs were weighed and measured. Out of a total of 25 parameters, only one, anteroposterior diameter of the proximal femur, was found to differ statistically between the bipedal and control groups. No changes were found in bone length, weight, or curvature. The results of other bipedal studies were reviewed, in light of these findings; and it is concluded that rats do not achieve true bipedalism, or increased hindlimb loading.

Craniofacial dysmorphogenesis following hypervitaminosis A in mice.

Malformations of the temporomandibular joint (TMJ), zygomatic arch, middle ear ossicles, and mandibular musculature following hypervitaminosis A were described in fetal mice. Pregnant mice (Mus musculus) were each given a 0.2-ml solution of corn oil containing 10,000 IU of retinol palmitate by gavage on day 8.7. Thirty-eight fetal heads were collected and hemisected. The left hemiheads were serially sectioned, stained, and examined histologically. Right halves were cleared and double-stained with alizarin red and alcian blue. The craniofacial morphologies of the test mice were found to vary from normal to maximally involved (feature unidentifiable) even among littermates. Both inter- and intralitter variation were discussed. Errors in chondrogenesis were determined to have produced the variety of dysmorphologies observed. Changes in Meckel's cartilage affected both the TMJ and ossicles; the presence of ectopic cartilages affected the zygomatic arch; and the musculature was affected secondary to skeletal system changes. Several modes of vitamin A interference leading to craniofacial dysmorphogenesis have been proposed in the literature. It was determined that proposals implicating altered cellular differentiation were the most compatible with the in vivo data recorded here and elsewhere.

A cinephotographic study of the role of the canine in limiting lateral jaw movement in Macaca fascicularis.

Three specimens of Macaca fascicularis were examined to test the hypothesis that the canines do not restrict lateral masticatory movements in these animals. The animals were filmed while chewing freely before, shortly after, and seven weeks after canine removal. Facial markers, viewed from the lateral plane, allowed for computer tracings of all mandibular movements. The maximal lateral movement of the mandible was assessed for each chewing cycle, and means were calculated for each test condition and animal. These means were compared intra-animal for statistically significant differences in lateral jaw movement before and after canine removal. No significant long-term increase in lateral movement was found in any animal after the removal of its canine teeth. We conclude that, in accordance with our hypothesis, the lateral movement during mastication did not increase significantly after removal of the canine teeth in three specimens of Macaca fascicularis. Therefore, the use of the macaque as a human model for masticatory studies requires further assessment. This study reveals that the canine teeth are not as important as previously assumed in affecting the form of the masticatory cycle in the macaque.

The phenotypic interdependence of the musculoskeletal characters of the mandibular arch in mice.

Evidence from studies of craniofacial anomalies and the evolutionary transition from reptiles to mammals suggests that the temporomandibular joint (TMJ), bony zygomatic arch, middle ear ossicles and mandibular muscle pattern may form a correlated suite of characters. To test the degree of phenotypic interdependence among these features, mandibular arch defects were analysed in prenatal mice. Retinoic palmitate was administered to pregnant mice on day 8.7 to produce test foetuses with malformations of the mandibular arch. A rating scale was developed for each of the four characters so that numerical values could be assigned to each phenotype encountered. Control animals were used to establish normal phenotypes for each character which were assigned a value of 1. Data from each test age, 16, 18 and 19 days postconception, were pooled and Spearmann rank correlation coefficients between each of the traits were calculated. Coefficients (R) range from a high of 0.87, between the TMJ and zygomatic arch, to a low of 0.67 between the zygomatic arch and the mandibular musculature showing highly significant correlations (P less than 0.0001) among all characters. Therefore, the data suggest that the musculoskeletal features of the mandibular arch are phenotypically interdependent during development.

Type I collagen and fibronectin synthesis by retrocorneal fibrous membrane.

The primary cultures obtained from the experimentally induced retrocorneal fibrous membrane synthesized and secreted into the medium mainly type I procollagen. This collagen was characterized after limited pepsin treatment and identified as type I collagen by the following criteria: (1) it contained two alpha 1 chains and one alpha 2 chain, (2) its sedimentation behavior was identical to that of type I collagen from skin, and (3) its peptide map after limited proteolysis with Staphylococcus aureus V8 protease was identical to that of type I collagen. The medium contained procollagen I, which was converted into alpha size chains by limited pepsin treatment, whereas the cellular fraction contained type I collagen already processed to its end product. Type III collagen and basement membrane collagen were present as minor components in this system Fibronectin, one of the major glycoproteins in extracellular matrices, was also synthesized and secreted into the medium. In contrast, normal corneal endothelial cells produce mainly basement membrane collagen.