Substrate ifluences human epidermal melanocyte attachment and sperading in vitro

Summary Previous culture systems for melanocytes have employed serum-supplemented medium and uncoated plastic dishes, prohibiting examination of possible substrate influences on cellular morphology and function. We now report, using a sensitive serum-free system and a quantitative procedure for evaluating cellular morphology, that modification of the plating surface affects human epidermal melanocyte attachment rate and subsequent morphology in vitro. Melanocytes attach and spread more rapidly on surfaces coated with fibronectin or Type I/III collagen or on surfaces previously conditioned by human keratinocytes, dermal fibroblasts, melanocytes, or melanoma cells than do melanocytes on untreated control surfaces. Type IV collagen and laminin, although minimally beneficial for cell attachment, do support a characteristics melanocyte morphology that differs from that seen either on the other coated surfaces or on uncoated plastic controls. Addition of fetal bovine serum at the time of inoculation has no appreciable effect on attachment but markedly improves cell spreading on untreated surfaces, while addition of nerve growth factor with or without serum to this system fails to affect cell attachment or spreading. Our data establish that human epidermal melanocytes are indeed capable of responding morphologically to substrate signals. The ability of several biochemically unrelated surfaces to enhance melanocyte attachment rate and spreading suggests that melanocytes have surface receptors with a variety of specificities. This work is relevant to the development of improved culture systems for melanocytes in vitro and to understanding melanocyte behavior in vivo. This work was supported by the USDA Agricultural Research Service, by a grant from Cheesebrough-Ponds, Inc., and by a Dermatology Foundation Fellowship (Dr. Yaar).     

CCN3 controls 3D spatial localization of melanocytes in the human skin through DDR1

Melanocytes reside within the basal layer of the human epidermis, where they attach to the basement membrane and replicate at a rate proportionate to that of keratinocytes, maintaining a lifelong stable ratio. In this study, we report that coculturing melanocytes with keratinocytes up-regulated CCN3, a matricellular protein that we subsequently found to be critical for the spatial localization of melanocytes to the basement membrane. CCN3 knockdown cells were dissociated either upward to the suprabasal layers of the epidermis or downward into the dermis. The overexpression of CCN3 increased adhesion to collagen type IV, the major component of the basement membrane. As the receptor responsible for CCN3-mediated melanocyte localization, we identified discoidin domain receptor 1 (DDR1), a receptor tyrosine kinase that acts as a collagen IV adhesion receptor. DDR1 knockdown decreased melanocyte adhesion to collagen IV and shifted melanocyte localization in a manner similar to CCN3 knockdown. These results demonstrate an intricate and necessary communication between keratinocytes and melanocytes in maintaining normal epidermal homeostasis.     

Extracellular matrix components influence the survival of adult cardiac myocytes in vitro

Calcium-tolerant myocytes were isolated from adult rat hearts by collagenase perfusion and plated on various substrates in serum-free medium and their adhesion to various extracellular matrix (ECM) components was determined. The myocytes attached readily to dishes coated with collagen type IV (C-IV), laminin (LN), and to fetal bovine serum (FBS) in a manner dependent on the concentration of the components. Substantially fewer myocytes adhered to dishes coated with fibronectin (FN) or to uncoated plastic dishes. Cells adhered equally well to dishes coated with C-IV, LN and FBS within 1-4 h. However, when examined after 2 weeks in culture it was found that only C-IV and LN could support survival of the attached myocytes, and when cultured on C-IV or LN the myocytes were spread and had formed a dense monolayer. The actin filaments had at this time reorganized linearly along the long axis of the cell and the myocytes contracted spontaneously. Rabbit antibodies were raised against myocyte membranes and their ability to inhibit attachment to ECM components was studied. Purified IgG inhibited attachment to C-IV, while having only a minor effect on attachment to LN. These data are compatible with the presence of a specific cell surface component(s) that interacts with ECM substrates and influences cell shape and possibly thereby influences cellular functions.     

Extracellular matrix modulates the function of human melanocytes but not melanoma cells

Normal human epidermal melanocytes are attached to a basement membrane, a specialized form of extracellular matrix (ECM), located between the epithelium and underlying dermal tissues. To determine whether ECM influences pigmented cell behavior in vitro, human epidermal melanocytes and melanoma cells were cultured on uncoated or ECM-coated plastic culture surfaces, and a comparison was made between growth and function in the presence or absence of ECM. Melanocytes cultured on ECM-coated surfaces developed flatter and larger cell bodies and produced more melanin than melanocytes cultured on uncoated surfaces. In the presence of phorbol-myristate-acetate and cholera toxin, the rate of melanocyte replication was increased by ECM. In the absence of these mitogens, ECM significantly enhanced the adhesiveness of nonproliferating melanocytes. ECM had little or no effect on these parameters (morphology, tyrosinase activity, replication) in a pigmented human malignant melanoma cell line. These findings indicate that normal human epidermal pigment cells have the ability to recognize and respond to matrix signals, whereas this capacity appears to be absent in melanoma cells.     

Thrombospondin-induced attachment and spreading of human squamous carcinoma cells

Thrombospondin (TSP) induced the attachment and spreading of human squamous carcinoma cells on plastic culture dishes and dishes coated with type I or type IV collagen. Increased adhesion was detected as early as 15 min after treatment. Dose-response studies indicated that 1-5 micrograms of TSP per 35 mm (diameter) culture dish was sufficient to induce a response and that a half-maximal response occurred at 10 micrograms of TSP/dish. The squamous carcinoma cells synthesized TSP as indicated by biosynthetic labeling experiments. TSP was secreted (or shed) into the culture medium by these cells and also became bound to the cell surface. TSP also promoted adhesion of human keratinocytes, fibroblasts and fibrosarcoma cells but did not induce attachment or spreading of human melanoma or glioma cells, although these cells did respond to laminin.     

Magnesium-dependent attachment and neurite outgrowth by PC12 cells on collagen and laminin substrata

We report a study of the substratum and medium requirements for attachment and neurite outgrowth by cells of the pheochromocytoma-derived PC12 line. In attachment medium containing both Ca2+ and Mg2+, more than 50% of cells attached within 1 hr to petri dishes coated with native collagen Types I/III or II, native or denatured collagen Type IV, laminin, wheat germ agglutinin (WGA), or poly-L-lysine; attachment to dishes coated with nerve growth factor (NGF) was only about 20% and attachment to uncoated dishes or to dishes coated with fibronectin or gelatin was almost nil. Neither prior culturing in the presence of NGF nor addition of NGF to the attachment medium significantly affected the extent of attachment to collagen or laminin. With Ca2+ (1 mM) as the sole divalent cation, cells attached normally to WGA, polylysine, and NGF, but failed to attach to collagen or laminin. With Mg2+ (1 mM) as the only divalent cation, attachment to all substrata was about the same as in medium with both Ca2+ and Mg2+. Like the ionic requirements, the kinetics of attachment, insensitivity to protease treatment of the cells, and inhibition by low temperature and sodium azide were similar for PC12 attachment to collagen and laminin, suggesting that a common molecular mechanism may underlie attachment to these substrata. The only significant difference observed was that addition of WGA (30 micrograms/ml) to the attachment medium inhibited attachment to collagen but promoted attachment to laminin. Finally, PC12 cells extended neurites on laminin, on native collagens I/III, II, and IV, and on denatured collagen IV; they did not extend neurites on denatured collagens I/III or II, NGF, or WGA. Neurite outgrowth on collagen and laminin occurred with Mg2+ as the sole divalent cation. These results suggest that the same Mg2+-dependent adhesion mechanism operates at the cell body and at the growth cone.     

Stimulation of Cultured Melanocytes in Medium Containing a Serum Substitute: Ultroser-G

Melanocyte cultures were established and maintained routinely in Ham's F-10 medium containing 12-O-tetradecanoyl-phorbol-13-acetate (TPA), isobutylmethylxanthine (IBMX), cholera toxin (CT) and fetal calf serum (FCS). Three serum substitutes (Ultroser-G, Nutridoma-Hu and Nutricyte-H) were tested in order to obtain a medium without FCS having a more constant composition. Melanocyte proliferation was examined in long-term culture experiments by in situ cell counts at different periods of time. Only with Ultroser-G (1-2%) was the proliferation of melanocytes maintained without both FCS and CT, whereas the addition of the other two serum substitutes resulted in stabilization of melanocyte densities in the cultures up to 28 days. In the medium containing 1% Ultroser-G and IBMX without TPA minimal or no increases in melanocyte density were found. Addition of basic fibroblast growth factor (bFGF, 1 ng/ml) to the medium without TPA resulted in a partial restimulation of growth in different experiments. In this system with 1% Ultroser-G and 1 ng/ml bFGF, IBMX could also be replaced by other factors (dbcAMP LTC4 and a purified form of α-melanocyte stimulating hormone). The culture medium with 1% Ultroser-G containing TPA and IBMX is now used for routine melanocyte culture. In this medium TPN/IBMX can easily be replaced by bFGF/dbcAMP with optimal growth stimulation. The combination bFGF/α-MSH and other more physiological stimulators offers an alternative to study responses of melanocytes in culture with respect to proliferation, metabolism, and phenotype.     

Protein Kinase C and Melanocyte Transformation

Melanoma cells which have been isolated from metastatic melanoma tissue are able to survive and proliferate in serum supplemented media. In contrast, normal human melanocytes require the presence of growth stimulators if they are to survive in culture. A tumor promotor, 12–0-tetradecanoyl-phorbol-13-acetate (TPA) and substances that increase intracellular levels of cyclic-adenosine-monophosphate (cAMP), such as cholera toxin or isobutylmethyl xanthine, have been widely used for this purpose. The phorbol diester receptor was shown in 1982 to be the phospholipid- and calcium-dependent enzyme protein kinase C (PKC). We therefore directed our studies to the role of PKC regulation in the growth of normal human melanocytes and their transformation. Our studies show that while melanoma cells are inhibited by TPA, the growth of normal melanocytes is stimulated in a dose-dependent manner. The inhibitor, 1-(5-isoquinolinesulfonyl)-2-methyl-piperizine dihydrochloride (H7), which has been found to be the most specific for PKC, had no effect on the growth of normal melanocytes, but inhibited the growth of melanoma cells in a dose-dependent manner. PKC was isolated from the membrane and cytosol of normal melanocytes and melanoma cells. The basal (resting) levels of PKC activity in normal melanocytes was low compared to that measured in melanoma cells, and after short-term (1 hour) treatment with TPA the PKC activity was greatest at the membrane, with the activity decreasing the cytosol. Upon prolonged (48 hours) treatment with TPA, this redistribution of activity continued in normal melanocytes and the total activity increased. In melanoma cells, however, the total PKC activity decreased, particularly in the membrane fraction. A difference in activity and distribution of the enzyme was also seen after short-term (1 hour) treatment with H7. There was very little effect seen on PKC in normal melanocytes; however, the effect on melanoma cells was similar to that seen after 48 hours of exposure to TPA with a decrease in total activity, particularly in the membrane fraction. These results indicate that the regulation of PKC, in particular its activation by TPA, is altered during the transformation of normal human melanocytes     

Growth characteristics of human epidermal melanocytes in pure culture with special reference to genetic differences

Human melanocyte cultures were established using disaggregated epidermal cell suspensions derived from foreskins and plated onto culture dishes in medium containing 2% fetal bovine serum, growth factors, hormones, and melanocyte growth factor (MGF) extracted from bovine hypothalamus (Wilkins et al., J.Cell. Physiol., 122:350, 1985). After 2 days in culture the cells were transferred to serum-free medium to eliminate keratinocyte and fibroblast growth. Melanocytes grew preferentially and pure melanocyte populations could be harvested after 12-16 days in vitro. Melanocytes were later subcultured in the presence of 1% FBS. Pure melanocyte cultures were characterized by light and electron microscopic criteria, as well as by cytochemical demonstration of the melanocyte-specific enzyme, tyrosinase. At the ultrastructural level, cultured melanocytes derived from black (negroid) neonatal skin (B-M) had numerous mature rod-shaped stage IV melanosomes, while white (caucasoid) skin-derived melanocytes (W-M) in culture contained no mature melanosomes. Growth rate, cell yield, and in vitro lifespan for B-M were more than twice that for W-M in pure melanocyte cultures in the presence of MGF. Our results suggest that MGF-dependent growth of B-M differs from that of W-M.     

Keratinocyte Extracellular Matrix-Mediated Regulation of Normal Human Melanocyte Functions

Active roles of cell-cell interaction between melanocytes and neighboring keratinocytes for the regulation of melanocyte functions in the skin have been suggested. We examined substantial regulatory mechanisms of keratinocyte extracellular matrix (kECMs) for normal human melanocyte functions without direct cell-cell contact. We specially devised kECMs from proliferating or differentiating keratinocytes and further treated them with environmental stimulus ultraviolet B (UVB) for skin pigmentary system. Normal human melanocytes (NHM) were cultured on the various keratinocyte ECMs and initially the effects of the kECMs upon melanocyte morphology (dendrite formation and extension), growth, melanin production and expressions of pigmentation-associated protein (MEL-5) and proliferation-associated protein (proliferating cell nuclear antigen; PCNA/cyclin) were studied. Then we compared the effects of these cell-matrix interactions with those of direct melanocyte-keratinocyte, cell-cell contact in co-culture on melanocyte functions. Melanocytes cultured on any types of the kECMs that were tested significantly extended dendrites more than that on plastic cell culture dish without kECM (control). Melanocytes cultured on the kECM prepared from UVB irradiated differentiating keratinocytes resulted in 219% increase in the number of dendrites. The growth of melanocytes on kECMs was also stimulated up to 280% of control. The kECM produced by proliferating keratinocytes had a more significant effect on the growth than kECM from differentiating keratinocytes. This melanocyte growth stimulating effect was decreased with kECM from UVB treated differentiating keratinocytes. The melanin content per melanocyte was constant on any of the kECMs. Expression of pigmentation-associated protein detected by monoclonal antibody, MEL-5, was not changed on the kECM, while it was increased in melanocytes in co-culture with keratinocytes. Expression of PCNA/cyclin in melanocytes cultured on kECMs was generally downregulated on kECM and in co-culture compared to that in a control culture. We demonstrated that the kECMs play important roles in the melanocyte morphology and proliferation. These observations suggest that environmental (UVB) and physiological (Ca⁺⁺) stimuli can regulate melanocyte functions through the keratinocyte extracellular matrix in vivo.     

Effects of biologically active tumour-promoting and non-promoting phorbol esters on in vitro growth of melanocytic cells

Sapintoxin A (SAP A), a naturally occurring biologically active but non-promoting phorbol ester, acts as an effective in vitro mitogen for freshly derived human melanocytes. Seven days after addition of 50 nM SAP A there was a four to fivefold increase in melanocyte number over that observed in untreated control cultures comparable to that achieved with a 50 nM concentration of 12-0-tetradecanoylphorbol 13-acetate (TPA). The fluorescent stage 2 promoter sapintoxin D (SAP D) also supported the growth of these cells, with a 50 nM dose producing an increase in cell number comparable to that observed with 200 nM TPA. Similar results were obtained with an established, but non-tumorigenic, line of murine melanocytes. The same compounds exerted a potent anti-proliferative effect against transformed melanocyte lines of murine and human origin associated with morphological alterations and an increase in melanin production consistent with induced cytodifferentiation.     

Hyaluronic acid inhibits adhesion of hepatic stellate cells in spite of its stimulation of DNA synthesis

Unbalanced accumulation of fibers in extracellular matrix (ECM) results from attachment and activation of hepatic stellate cells (HSCs) during chronic liver diseases, in which the content of hyaluronic acid (HA), a glycosaminoglycan, in ECM changes. No information is available on the effect of HA on adhesion and activation of HSCs although that of collagen (Col) on HSCs was extensively studied. This study investigated the effects of HA with or without Col on adhesion of HSCs or the rate of DNA synthesis. Attachment of primary cultured HSCs was microscopically monitored in the plate simultaneously coated with HA or other ECM components. HA inhibited adhesion of quiescent HSCs at least up to 7 days after seeding, whereas HSCs were adherent to plastic or type I collagen (Col-I), type III collagen (Col-III), type IV collagen (Col-IV) or fibronectin. Both microscopy and alpha-smooth muscle actin immunocytochemistry revealed that the number of HSCs, which had been re-seeded after 15 days of culture, attached to HA-coated area was remarkably lower compared to that of HSCs on Col-I or plastic. Incorporation of HA into Col-I prevented adhesion of activated HSCs to matrix film. The number of HSCs adherent to HA at early times after seeding was minimal and significantly lower than that of the cells adherent to plastic. In contrast, either Col-I or Col-IV increased the number of adherent cells. Attachment of HSCs to plastic was inhibited by soluble HA in culture medium. CD44, the cell surface receptor to which HA binds, was immunochemically detected in HSCs. Adhesion of HSCs to plastic, HA or Col-I was not changed by anti-CD44 antibody. Either HA or Col increased the basal or platelet-derived growth factor-inducible rate of thymidine incorporation into DNA in HSCs. In conclusion, HA inhibits adhesion of quiescent or activated HSCs in spite of its stimulation of DNA synthesis, whereas Col increases HSC attachment and DNA synthesis, and inhibition of HSC adhesion by HA does not involve CD44.     

Endothelin-1 combined with extracellular matrix proteins promotes the adhesion and chemotaxis of amelanotic melanocytes from human hair follicles in vitro

During the repigmentation of vitiliginous skin, amelanotic melanocytes (AMMCs) migrate from the outer root sheath (ORS) of the hair follicles into depigmented skin. It has been shown that endothelin-1 (ET-1) is an important cytokine in the migration of epidermal melanocytes, produced by keratinocytes particularly after irradiation with ultraviolet B (UVB). To further examine the role of ET-1 on the migration of AMMCs, we investigated the effects of ET-1 to the adhesion and chemotaxis of human AMMCs combined with extracellular matrix proteins (ECMP) and observed the effects on the actin and tubulin cytoskeleton of AMMC by ET-1. Human AMMCs were treated with different concentrations of ET-1 (0.1-100 nM) to observe adhesion on culture dishes coated with fibronectin (FN), laminin (LN) and collagen IV (CIV). In addition, chemotaxis on FN, LN and CIV coated micropore filters, with various concentrations of ET-1 as attractants, was investigated using a Boyden chemotaxis chamber. Cellular microfilaments and microtubules were immunostained with Rhodamine labeled actin and FITC labeled beta-tubulin. The effects of ET-1 on cytoskeleton were observed with laser confocal microscopy. The study demonstrated that ET-1 increases human AMMCs adhesion on FN in a dose-dependent manner, but minor increases are found on the coated surface with LN and CIV. ET-1 also induces chemotaxis of AMMCs on CIV, LN and FN in a dose-dependent manner. The greatest effect was seen with CIV. Minor chemotactic effects were observed with non-coated surfaces. A concentration of >or=10nM ET-1 induced an apparent increase in stress fibers underneath the cell membrane, but no effects were found on tubulin. ET-1 has various effects on the adhesion and chemotaxis of AMMCs on various ECMP, which could be partly due to a modulation and reorganization of the actin cytoskeleton.     

Inhibition of cell adhesion by type V collagen.

Human umbilical vein endothelial cells grew well in dishes coated with collagen types I, II, III, or IV. However, the same cells tended to detach themselves from dishes coated with type V collagen, and cell proliferation in these dishes was inhibited. Such anti-adhesive activity was partially retained by heat-denatured type V collagen or by its alpha 1 chain, but not by its alpha 2 chain. Several other cell types did not adhere to the type V collagen substratum even in the presence of 10% serum. The cell types strongly inhibited from adhering by type V collagen included Swiss mouse 3T3 cells and their MSV-transformants, BALB/c 3T3 cells and their methylcholanthrene-transformants, NIH 3T3 cells and their ras-transformants, BHK cells, CHO-9 cells, CHO-K1 cells, and mouse melanoma B16-F10 cells. Using Swiss mouse 3T3, we studied the effects of type V collagen on cell adhesion to fibronectin in serum-free medium. When the culture dishes were coated with a mixture of fibronectin with various concentrations of type V collagen, the adhesion of the cells was inhibited depending on the concentration of type V collagen. The inhibition of cell adhesion by type V collagen was competitively overcome by increased concentrations of fibronectin. The activity that interferes with the effects of fibronectin was retained mainly by the alpha 1 chain of heat-denatured type V collagen.     

Effect of serum,fibronectin, and laminin on adhesion of rabbit intestinal epithelial cells in culture

Rabbit intestinal epithelial cells, obtained after a limited hyaluronidase digestion, were incubated in medium with or without calf serum, on bacteriological plastic dishes. The dishes, either plain or coated with an air-dried type I collagen film, were pretreated with medium alone or with medium containing purified laminin or purified fibronectin. Cells did not attach in significant numbers to untreated bacteriological plastic, even in the presence of serum. Cells did attach to collagen-coated dishes, and were judged viable on the basis of their incorporation of radiolabeled leucine into cell protein. Cell adhesion to the collagen substrate increased in proportion to the concentration of serum in the medium, with maximal attachment at 5% serum or greater. Pretreatment of plain or collagen-coated dishes with increasing amounts of fibronectin enhanced cell adhesion in a concentration-dependent manner. Either serum, or fibronectin-free serum in the medium enhanced cell attachment to substrates pretreated with cither fibronectin or laminin. Thus, intestinal epithelial cells appear to possess surface receptors for both laminin and fibronectin. The evidence further suggests that calf serum may contain factors, other than fibronectin, capable of enhancing intestinal epithelial cell attachment to collagen substrates.     

PC12 adhesion and neurite formation on selected substrates are inhibited by some glycosaminoglycans and a fibronectin-derived tetrapeptide

The effects of added soluble glycosaminoglycans (GAGs) on adhesion and neurite formation by cultured PC12 pheochromocytoma cells on several substrates were tested. PC12 cells adhere more rapidly to Petri plastic coated with fibronectin, laminin, poly-L-lysine, or conA, than to either uncoated Petri plastic or tissue culture plastic. Adhesion to poly-L-lysine, fibronectin- and laminin-coated dishes was significantly inhibited by added dextran sulfate and to a lesser extent heparin--but not by chondroitin sulfate. PC12 adhesion to fibronectin could also be totally inhibited by the putative fibronectin cell binding tetrapeptide L-arginyl-glycyl-L-aspartyl-L-serine (Pierschbacher, MD & Ruoslahti, E, Nature 309 (1984) 30). The inhibitory effects of combinations of this tetrapeptide and heparin or dextran sulfate (but not chondroitin sulfate or hyaluronic acid) were additive. Nerve growth factor (NGF) pretreatment increased the percentage of PC12 cells adherent to all substrates and reduced the GAG inhibition of adhesion. PC12 cells previously treated with NGF to induce morphologic differentiation will rapidly re-extend neurites when plated on all four substrates. On fibronectin and poly-L-lysine-coated dishes this neurite growth is inhibited by added heparin and dextran sulfate, while on laminin it is not. Neurite formation on fibronectin-coated dishes was also inhibited by low concentrations of fibronectin tetrapeptide. In summary, PC12 adhesion and neurite formation can be inhibited by sulfated GAGs on some substrates, including fibronectin, but not other substrates, suggesting that these cells have at least two independent molecular adhesion mechanisms.     

Intercellular contacts and the organization of actin filaments in cultured epithelial FL cells are altered by growth on type I collagen and treatment with 12-O-tetradecanoylphorbol-13-acetate through the modulation of interactions between cells and the substratum

We have investigated the effects of various types of collagen and a tumor-promoting phorbol ester on intercellular contacts and the organization of actin in human amnion epithelial FL cells and mouse fibroblast 3T3-A31 cells. Our purpose was to investigate how modulation of interactions between cells and the substratum leads to alterations in intercellular contacts and organization of actin filaments. When cells were cultured on dishes coated with a solution containing type I collagen, but not type IV, changes were induced in the morphology of FL cells and their intercellular contacts. Type I collagen also caused changes in the organization of their actin filaments, although no such effects were observed with 3T3-A31 cells. In contrast, 12-O-tetradecanoylphorbol-13-acetate (TPA) caused morphological changes, dissociation of groups of cells, and reorganization of actin filaments in cultures of FL and 3T3-A31 cells. It also disrupted the sites of adhesion of FL cells to the substratum. Both type I collagen and TPA rapidly induced spreading of FL cells in the absence of serum. However, cis-hydroxyproline, known to inhibit secretion of collagen, did not suppress the TPA-induced dissociation of groups of FL cells. These results suggest that the interactions with type I collagen of epithelial FL cells, but not of fibroblastic 3T3-A31 cells, tend to disorganize cellular morphology, intercellular contacts, and actin filaments in ways similar to, but not directly related to, the effects of TPA.     

Adhesiveness and proliferation of epithelial cells are differentially modulated by activation and inhibition of protein kinase C in a substratum-dependent manner

In the present study, we have examined the regulation of attachment, onset of proliferation and the subsequent growth, in vitro, of chick retinal pigmented epithelial (RPE) cells as a function of the nature of the substratum and of either the activation or inhibition of protein kinase C (PKC). The RPE cells have an adhesive preference for protein carpets which contain laminin. This preference disappears gradually with time in culture. The adhesion of RPE cells to fibronectin is shown to be a receptor-mediated process which involves the RGD recognition signal. This study also demonstrates that a PKC activator, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), affects RPE cell adhesion in a substratum-dependent manner. Exposure of RPE cells to TPA lowers the cell attachment efficacy to ECM protein substrata but does not affect cell attachment to plastic. The onset of cell proliferation is accelerated by TPA on all of the substrata tested. The minimal duration of an effective TPA pulse exerting a long-lasting influence on RPE cell proliferation is between 1.5 and 3.5 hr. Stimulation of cell proliferation by TPA in long-term cultures is independent of the nature of the growth substratum. The acceleration of the onset of cell proliferation by TPA is sensitive to 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7), an inhibitor of conventional PKC, and thus appears to be dependent on the activation of conventional PKC. H7 also affects cell-cell contacts, causing an alteration in the shape (“squaring”) of RPE cells packed into large colonies. Conversely, the effects of TPA on both the attachment and the long-term proliferation of RPE cells are not dependent a conventional PKC isotype, since H7 cannot abolish the influence of TPA on either process. We conclude that the effect of TPA on long-term proliferation of RPE cells is either dependent on a novel PKC isotype or independent of PKC. © 1993 Wiley-Liss, Inc.     

Influence of macrophages and macrophage-modified collagen I on the adhesion and proliferation of vascular smooth muscle cells in culture

The adhesion, proliferation and morphology of rat vascular smooth muscle cells (VSMC) in cocultures with macrophages or in cultures on type I collagen modified by activated macrophages were evaluated. In the first set of experiments, rat alveolar macrophages were added to 24-hour-old VSMC cultures. Between days 2 and 5 after VSMC seeding, the population densities and doubling times of cells were similar in both VSMC-macrophage and pure VSMC cultures. However, from day 5, the cocultures proliferated about two times more rapidly and on day 7, they reached higher cell population density by 40%. The pure macrophage cultures did not proliferate. In the second set of experiments, rat alveolar macrophages were activated by non-toxic TiO2 dust to produce reactive oxygen species and incubated for 120 min with collagen I. The collagen was then adsorbed on plastic culture dishes and seeded with VSMC. The collagen exposed for 10 min only, the unmodified collagen and pure culture dishes were used as control growth supports. On all four tested substrates, the number of initially adhered cells was similar, but on the collagen modified for 120 min, the cells were less spread. Moreover, on day 2 to 3 after seeding, some cells on this collagen became vacuolated and detached spontaneously from the growth support. The remaining VSMC, however, rapidly proliferated, so that on day 9, the cell population density on 120-min-modified collagen was similar as on both control collagens and significantly higher compared to that on uncoated dishes. Our results suggest that 1. The delayed growth-stimulating effect of macrophages on VSMC-macrophage mixed population is probably due to autocrine production of mitogens by both cell types rather than due to an acute effect of short-living oxygen radicals released from macrophages immediately after adding to VSMC cultures. 2. The effect of collagen I exposed to activated macrophages for 120 min is slightly cytotoxic, which could, however, stimulate a release of mitogens from damaged as well as surviving VSMC.