Cellular and hormonal regulation of pigmentation in human ocular melanocytes.

The purpose of this study was to examine some of the factors that may be relevant to regulating pigmentation in the human eye, specifically whether choroidal and iridial melanocytes are sensitive to regulation by epithelial and stromal cells and alpha-melanocyte stimulating hormone (alpha-MSH). Human choroidal and iridial melanocytes were established in culture and co-cultured with epithelial cells and stromal cells derived both from skin and from eye in order to determine their influence on choroidal and iridial melanocyte dopa oxidase activity. In all cases, co-culture of melanocytes with either epithelial cells or fibroblasts led to an increase in dopa oxidase activity during 5 days of co-culture. The extent of the increase ranged from 60% (non-significant) to as much as 185% when both fibroblasts and keratinocytes were present. The optimal ratio of fibroblasts to melanocytes was 1:10 (for dermal fibroblasts) or 1:2 (for iridial fibroblasts) and 1:1 for all epithelial cells to melanocytes. Both choroidal (three out of three cultures) and iridial (two out of three cultures) melanocytes showed increases in dopa oxidase activity to alpha-MSH when cultured in Green's media but the same cells cultured in MCDB153 were unresponsive to alpha-MSH. These in vitro studies suggest that ocular melanocytes have the capacity to be influenced by adjacent epithelial and stromal cells with respect to pigmentation.

Influence of pigment content, intracellular calcium and cyclic AMP on the ability of human retinal pigment epithelial cells to contract collagen gels.

PURPOSE: The aim of the study was to determine to what extent collagen gel contraction could be reduced by calcium and calmodulin antagonists and agents that elevate cyclic AMP in order to develop a pharmacological approach to prevent/arrest RPE contraction of epiretinal membranes in proliferative vitreoretinopathy. We also explored a possible role of pigment in collagen gel contraction. METHOD: We measured RPE mediated contraction of 3D collagen gels in the presence and absence of the calcium and calmodulin antagonists TMB8, Verapamil and Tamoxifen and the cAMP elevating agents IBMX and Forskolin. The effect of pigment on collagen gel contraction was assessed by comparing gel contraction mediated by RPE cells re-pigmented with melanin with that mediated by unpigmented RPE. The effect of IBMX on RPE proliferation was assessed using a BrdU ELISA and the effects of IBMX on RPE cytoskeleton and cell shape were assessed using Actin and Cytokeratin immunocytochemistry. RESULTS: We report that both cAMP elevating agents and calcium and calmodulin antagonists reduce RPE mediated collagen gel contraction. Cyclic AMP elevation was more effective than a reduction in calcium in reducing contraction. There were no significant advantages in combining both approaches. The presence of melanin had no effect on gel contraction. Calcium antagonists and particularly agents which elevate cAMP caused RPE cells in collagen gels to extend fewer and shorter processes. cAMP elevation in particular caused RPE cells to become more rounded and develop arborized cell processes. Immunostaining for actin and cytokeratin revealed changes in cytoskeletal organisation in response to IBMX in that cells contained less actin than untreated cells and concentrated cytokeratins more centrally. CONCLUSION: We have identified two possible pharmacological approaches which may provide a new direction for preventing or slowing down the development of PVR.

Increased axon growth through astrocyte cell lines transfected with urokinase.

The ability of cells to migrate through tissues depends on their production of a variety of proteases, and the same may be true of growth cones. Urokinase (plasminogen activator) regulates much of the extracellular proteolytic activity, by activating other proteases and as a result of its own proteolytic activity. In order to evaluate the potential role of urokinase as a promoter of axon growth, we have used a plasmid expressing urokinase under a cytomegalovirus promoter to transfect an astrocyte cell line, Neu7, which we have previously shown to provide a poor environment for axon regeneration. Five transfected lines all showed greatly increased ability to promote axon regeneration in both monolayer and three-dimensional cultures. The critical change in the transfected cells was largely within the extracellular matrix, since extracellular matrix laid down by urokinase-secreting cells was more permissive to axon growth than matrix from the parent Neu7 line. The effect was due to urokinase since treatment of the transfected cells with the urokinase inhibitors B623 and B428 rendered both the cells and their matrix much less permissive to axon growth, but did not require plasminogen, since it was blocked neither by serum-free medium nor by plasmin inhibitors.

An analysis of astrocytic cell lines with different abilities to promote axon growth.

The adult mammalian central nervous system (CNS) lacks the capacity to support axonal regeneration. There is increasing evidence to suggest that astrocytes, the major glial population in the CNS, may possess both axon-growth promoting and axon-growth inhibitory properties and the latter may contribute to the poor regenerative capacity of the CNS. In order to examine the molecular differences between axon-growth permissive and axon-growth inhibitory astrocytes, a panel of astrocyte cell lines exhibiting a range of axon-growth promoting properties was generated and analysed. No clear correlation was found between the axon-growth promoting properties of these astrocyte cell lines with: (i) the expression of known neurite-outgrowth promoting molecules such as laminin, fibronectin and N-cadherin; (ii) the expression of known inhibitory molecules such tenascin and chondroitin sulphate proteoglycan; (iii) plasminogen activator and plasminogen activator inhibitor activity; and (iv) growth cone collapsing activity. EM studies on aggregates formed from astrocyte cell lines, however, revealed the presence of an abundance of extracellular matrix material associated with the more inhibitory astrocyte cell lines. When matrix deposited by astrocyte cell lines was assessed for axon-growth promoting activity, matrix from permissive lines was found to be a good substrate, whereas matrix from the inhibitory astrocyte lines was a poor substrate for neuritic growth. Our findings, taken together, suggest that the functional differences between the permissive and the inhibitory astrocyte cell lines reside largely with the ECM.

Increased axon regeneration in astrocytes grown in the presence of proteoglycan synthesis inhibitors.

We have recently reported that the critical difference between astrocytic cell lines that are good or poor promoters of axon growth lies in the extracellular matrix. We demonstrated that much of this difference between matrix produced by permissive and non-permissive cell lines could be ascribed to one or more dermatan/keratan sulphate proteoglycans and that these proteoglycans are able to block the neurite-promoting effect of laminin. These proteoglycans are also produced by cultures of primary astrocytes. In the present study, we have demonstrated that treatment of both astrocytic cell lines and primary astrocytes with inhibitors of proteoglycan synthesis, beta-D-xylosides and sodium chlorate, can strongly influence the axon growth promoting properties of both matrix and whole cells. Dorsal root ganglia grown on matrix or in conditioned medium from cultures treated with beta-D-xylosides or sodium chlorate had twice as many axons and the axons grew to twice the length as in control cultures. Following treatment of Neu7 cells with proteoglycan synthesis inhibitors there was also a significant reduction in the ability of Neu7 conditioned medium to block the neurite-promoting effect of laminin. Dorsal root ganglia grown on Neu7 cells treated with sodium chlorate extended 2 to 3 times the number of axons for approximately 300 mm longer distance than on control cultures. Treatment of Neu7 cells with beta-D-xylosides, however, did not make the cells less inhibitory to axon growth. We have also examined the effects of proteoglycan synthesis inhibitors on three-dimensional primary astrocyte cultures, which closely mimic the in vivo effects of astrocytes on axon growth.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein ubiquitination and neuronal differentiation in chick embryos.

Ubiquitin is a small highly conserved intracellular protein which is involved in a number of cellular functions including targeting proteins for degradation. We have studied the distribution of ubiquitin-protein conjugates and two enzymes involved in protein ubiquitination in chick embryos. Using immunocytochemical techniques, we have observed that chick neural crest cells and dorsal root ganglia acquire immunoreactivity in their nuclei and cytoplasm as they mature, both in vivo and in vitro, though they are not immunoreactive at early stages of development. Immunoreactivity for the ubiquitin activating enzyme (E1) and a carboxyl terminal hydrolase for ubiquitin (PGP 9.5) also appears in the nuclei of differentiating neurons at the same time as ubiquitin-protein conjugates. Our results provide evidence that the nuclear accumulation of ubiquitin-protein conjugates is closely associated with maturation of neurons towards a differentiated phenotype.

An inhibitor of neurite outgrowth produced by astrocytes.

We have produced a number of astrocytic cell lines, some of which promote abundant neurite outgrowth, some of which are poor promoters of neurite outgrowth. The critical difference between these lines lies in the extracellular matrix, cell lines that are good promoters of axon growth producing a matrix that promotes axon growth, cell lines that are poor promoters of axon growth producing a non-permissive matrix. We were unable to find any consistent correlations between promotion of axon growth and production of proteases, protease inhibitors, N-cadherin, growth cone collapsing activity, and several extracellular matrix molecules. In the present study we have compared the least permissive of our cell lines, Neu7, with the most permissive, A7. Medium conditioned by the cell lines has the same properties as the matrix, since dorsal root ganglia (DRGs) grown in conditioned medium from the Neu7 line grow axons poorly, while DRGs grown in medium conditioned by A7 or primary astrocytes grow many long axons. Since matrix produced by all the cell lines contains large amounts of laminin, we looked to see whether the cells were producing laminin-blocking activity. Medium from the Neu7 line blocked laminin, while that from the A7 and primary astrocytes did not. However, when the conditioned media were heat-treated to remove neurite-promoting activity, they all had laminin-blocking activity: the blocking activity is heat stable. The neurite-promoting properties of the conditioned media therefore probably reflect a balance between promoting molecules and blockers.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of embryonic retinal neurons on neural crest cell differentiation into Schwann cells.

Amongst the many cell types that differentiate from migratory neural crest cells are the Schwann cells of the peripheral nervous system. While it has been demonstrated that Schwann cells will not fully differentiate unless in contact with neurons, the factors that cause neural crest cells to enter the differentiative pathway that leads to Schwann cells are unknown. In a previous paper (Development 105: 251, 1989), we have demonstrated that a proportion of morphologically undifferentiated neural crest cells express the Schwann cell markers 217c and NGF receptor, and later, as they acquire the bipolar morphology typical of Schwann cells in culture, express S-100 and laminin. In the present study, we have grown axons from embryonic retina on neural crest cultures to see whether this has an effect on the differentiation of neural crest cells into Schwann cells. After 4 to 6 days of co-culture, many more cells had acquired bipolar morphology and S-100 staining than in controls with no retinal explant, and most of these cells were within 200 microns of an axon, though not necessarily in contact with axons. However, the number of cells expressing the earliest Schwann cell markers 217c and NGF receptor was not affected by the presence of axons. We conclude that axons produce a factor, which is probably diffusible, and which makes immature Schwann cells differentiate. The factor does not, however, influence the entry of neural crest cells into the earliest stages of the Schwann cell differentiative pathway.

Expression of Schwann cell markers by mammalian neural crest cells in vitro.

During embryonic development, neural crest cells differentiate into a wide variety of cell types including Schwann cells of the peripheral nervous system. In order to establish when neural crest cells first start to express a Schwann cell phenotype immunocytochemical techniques were used to examine rat premigratory neural crest cell cultures for the presence of Schwann cell markers. Cultures were fixed for immunocytochemistry after culture periods ranging from 1 to 24 days. Neural crest cells were identified by their morphology and any neural tube cells remaining in the cultures were identified by their epithelial morphology and immunocytochemically. As early as 1 to 2 days in culture, approximately one third of the neural crest cells stained with m217c, a monoclonal antibody that appears to recognize the same antigen as rat neural antigen-1 (RAN-1). A similar proportion of cells were immunoreactive in cultures stained with 192-IgG, a monoclonal antibody that recognizes the rat nerve growth factor receptor. The number of immunoreactive cells increased with time in culture. After 16 days in culture, nests of cells, many of which had a bipolar morphology, were present in the area previously occupied by neural crest cells. The cells in the nests were often associated with neurons and were immunoreactive for m217c, 192-IgG and antibody to S-100 protein and laminin, indicating that the cells were Schwann cells. At all culture periods examined, neural crest cells did not express glial fibrillary acidic protein. These results demonstrate that cultured premigratory neural crest cells express early Schwann cell markers and that some of these cells differentiate into Schwann cells. These observations suggest that some neural crest cells in vivo may be committed to forming Schwann cells and will do so provided that they then proceed to encounter the correct environmental cues during embryonic development.

The gut supports neurogenic differentiation of periocular mesenchyme, a chondrogenic neural crest-derived cell population.

Periocular mesenchyme (PM) is a mesencephalic neural crest derived cell population which as a result of an interaction with the retinal pigment epithelium forms the scleral cartilage of the avian eye. Enteric neurons are derived from vagal crest cells which invade the gut. To study factors which regulate neuronal differentiation, we investigated whether the gut could direct neurogenesis in PM, a cell population that does not produce neurons in vivo. We report here that PM cultured in the presence of aneural chick hindgut on the chorioallantoic membrane (CAM), invaded the gut and formed large numbers of neurons. These were localized in enteric ganglia and contained neurofilament immunoreactivity, vasoactive intestinal peptide immunoreactivity, and somatostatin immunoreactivity. In the control PM cultured alone on the CAM, a small number of cells contained neurofilament immunoreactivity but lacked the appearance of mature neurons.