The competence of transformed keratinocytes to differentiate is accompanied by amplification of the LDL- and EGF-receptor genes but not of the insulin receptor gene

The possible relationship between cell surface receptor numbers, receptor gene expression for low density lipoprotein (LDL), insulin and epidermal growth factor (EGF), and differentiation capacity has been studied in normal and SV40 transformed (SVK14) keratinocytes, various squamous carcinoma cell (SCC) lines and A431 cells. Our recent studies demonstrated that an inverse relationship exists between LDL- and EGF-receptor binding and the ability to differentiate of both normal and transformed keratinocytes. In the present study cloned LDL- and EGF-receptor complementary DNAs were used as probes to identify both LDL and EGF receptor gene fragments on genomic DNA blots. The extent of hybridisation was found to be increased to the highest extent in A431 cells and decreased in other cells in the following order SCC-4 greater than SCC-15. In SCC-12F2, SVK14 and normal keratinocytes no increase has been observed. The increased hybridisation of LDL- and EGF-receptors in A431, SCC-4 and SCC-15 cells was found to be due to gene amplification and not to aneuploidy. In contrast to the LDL- and EGF-receptor binding, no correlation has been found between insulin receptor binding and ability of cells to differentiate. Furthermore, no amplification of insulin receptor gene has been observed in any of the cells under study.     

Epidermal growth factor receptor expression related to differentiation capacity in normal and transformed keratinocytes

Epidermal growth factor (EGF) and Ca2+ have been indicated to play a major role in skin development. We have used normal keratinocytes, SV40-transformed keratinocytes (SVK14) and various squamous carcinoma cell (SCC) lines as in vitro model system to study the effect of the extracellular Ca2+ concentration of EGF-receptor expression in relation to the capability of cells to differentiate. The cell lines used exhibit a decreasing capacity to differentiate in the order of keratinocytes approximately SVK14 greater than SCC-12F2 greater than SCC-15 greater than SCC-12B2 greater than SCC-4, as judged from Ca2+-ionophore-induced cornified envelope formation. Under normal Ca2+ conditions, all cell lines (except for SCC-15) exhibited two classes of EGF-binding sites. The number of low-affinity binding sites increased considerably as cells were less able to differentiate, while the apparent dissociation constant (kd) was similar in all cell lines. In contrast, the properties of high-affinity EGF binding varied in the various cell lines without a clear relationship to the degree of differentiation capacity. Lowering the extracellular Ca2+ concentration to 0.06 mM resulted in a decrease of Ca2+ ionophore-induced cornified envelope formation, demonstrating the decreased ability to differentiate under these conditions. The decreased ability to differentiate was accompanied by a marked increase in the number of EGF-binding sites, but without a change of the kd. Furthermore, no high-affinity EGF-binding sites were detectable under these conditions. Finally, addition of Ca2+ to low Ca2+-cultured cells caused a rapid decrease of EGF binding in all cell lines, most prominently in normal keratinocytes and SCC-12F2 cells. The data presented demonstrate: The combination of normal keratinocytes, SVK14 and the various SCC lines provides an attractive model system to study differentiation in vitro; EGF-receptor expression is related to the state of differentiation, both phenomena being sensitive to the external Ca2+ concentration; and EGF-receptor expression is related to the capability of cells to differentiate.     

Phorbol ester binding and protein kinase C activity in normal and transformed human keratinocytes

Normal keratinocytes, SV40-transformed keratinocytes (SVK14), and various squamous carcinoma cell (SCC) lines have been used as an in vitro model system to study the properties of phorbol ester receptor and protein kinase C expression during keratinocyte differentiation. The cell lines used exhibit a decreasing capacity to differentiate in the order of keratinocytes approximately SVK14 greater than SCC-12F2 greater than SCC-15 greater than SCC-4; moreover, all cell lines respond to a low external Ca2+ concentration by a decreased capacity to differentiate. Normal keratinocytes exhibited the highest number of phorbol ester receptors as compared to the other cell lines, while each individual cell line exhibited a higher number of phorbol ester receptors during growth under normal Ca2+ conditions as compared to cells grown under low Ca2+ conditions. The apparent dissociation constant (Kd) demonstrated only small variations in the various cell lines. In contrast, the cytoplasmic protein kinase C activity, was found to be higher in cells grown under low Ca2+ conditions than in cells grown under normal Ca2+ conditions, indicating the absence of a causal relationship between cytoplasmic protein kinase C activity and phorbol ester receptor expression. Therefore the properties of protein kinase C have been determined in more detail in normal keratinocytes and SCC-15 cells. These studies revealed differences between protein kinase C properties from the two cell lines grown under normal and low Ca2+ conditions. The differences included the effect of phorbol 12-myristate 13-acetate (PMA) on the redistribution pattern of protein kinase C between the cytoplasmic and particulate fractions as well as the activating effect of diolein in vitro on protein kinase C activity, partly purified from particulate or cytoplasmic fractions. These observations demonstrate that the functional protein kinase C activity of keratinocytes is determined by various endogenous and exogenous activators and that these activators are modulated differently in various cell lines, under various growth conditions (low Ca2+ versus normal Ca2+).     

Modulation of low density lipoprotein receptor activity in squamous carcinoma cells by variation in cell density

Morphological and biochemical studies on low density lipoprotein (LDL) receptor metabolism were performed in squamous carcinoma cells (SCC-15 and SCC-12F2). Modulation of terminal differentiation was achieved by culturing these cells at different cell densities. Studies on these cells cultured at low density (hardly any terminal differentiation) showed the following results: High affinity binding of LDL was excessive; LDL binding to SCC-15 cells was twice as high as that in SCC-12F2 cells and in fibroblasts. The distribution of the LDL binding visualized by LDL receptor antibodies was non-linear. There was no contact inhibition of LDL binding. LDL-gold particles were mainly bound to the plasma membrane outside coated pits. LDL-gold particles were internalized and delivered to dense bodies (= lysosomes). Degradation of LDL took place after a lag period of 10 min. Dissociation of LDL from the plasma membrane was substantial (more than 40% after a 120 min chase period). The same experiments on the cells cultured at high density (terminal differentiation present) showed several differences: A sharp decrease in high affinity LDL binding in both cell types. The internalization of surface bound LDL was defective in most of the squamous carcinoma cells. Dissociation of LDL from the plasma membrane was substantial, and after a chase period of 120 min at 37 degrees C still more than 20% of LDL remained intracellular and was not degraded. We postulate that LDL receptor-mediated endocytosis and degradation take place in squamous carcinoma cells but that during the process of terminal differentiation modulation of LDL-receptor metabolism occurs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of retinoids on differentiation, lipid metabolism, epidermal growth factor, and low-density lipoprotein binding in squamous carcinoma cells

The relationship among keratinocyte differentiation capacity, lipid synthesis, low-density lipoprotein (LDL) metabolism, plasma membrane composition, and epidermal growth factor (EGF) binding has been studied in SCC-12F2 cells. The differentiation capacity of the cells, i.e., ionophore-induced cornified envelope formation, was inhibited by various retinoids and stimulated by hydrocortisone. Retinoids that caused a significant reduction of cornified envelope formation, i.e., retinoic acid and 13-cis-retinoic acid, caused only minor changes in lipid synthesis and plasma membrane composition. Arotinoid ethylsulfone, having a minor effect on cornified envelope formation, caused a drastic inhibition of cholesterol synthesis, resulting in changes in the plasma membrane composition. Hydrocortisone stimulated cornified envelope formation but had only minor effects on lipid synthesis and plasma membrane composition. Of all retinoids tested, only arotinoid ethylsulfone caused a drastic increase in EGF binding, while hydrocortisone had no effect. Retinoic acid, arotinoid ethylsulfone, and hydrocortisone had no effects on LDL binding and only minor effects on LDL degradation. These results clearly demonstrate that the plasma membrane composition is not related to keratinocyte differentiation capacity, but most likely does determine EGF binding. Furthermore, EGF binding does not determine keratinocyte differentiation capacity.     

Calcium-mediated regulation of the low density lipoprotein receptor and intracellular cholesterol synthesis in human epidermal keratinocytes

Unlike cells cultured under physiological Ca2+ concentrations (1-2 mM), keratinocytes cultured in media containing Ca2+ in low concentrations (less than 0.1 mM) do not stratify. The latter cells also differ with respect to several features of the regulation of cholesterol synthesis. In keratinocytes cultured in medium containing high Ca2+ concentrations (1.6 mM) and fetal calf serum, the rate of cholesterol synthesis was 20-30 times higher than in keratinocytes exposed to a low Ca2+ concentration. The rate of cholesterol synthesis did not change when high-calcium cells were deprived of extracellular sources of cholesterol but increased (8-10 fold) in deprived low-calcium cells. Furthermore, the addition of low density lipoprotein (LDL) reduced cholesterol synthesis markedly in low-calcium cells but had no effect on high-calcium cells. Finally, in keratinocytes cultured at low calcium concentrations the association and degradation of 125I-LDL was 20-30 times higher than in keratinocytes cultured under high-calcium conditions. Switching of the cells from the low-calcium to the high-calcium medium resulted in the induction of terminal differentiation within 15 hours and was accompanied by increased cholesterol and protein synthesis, increased competence of cells to form cornified envelopes, and reduced association of 125I-LDL. A gradual increase of the extracellular Ca2+ concentration was accompanied by a corresponding increase of cholesterol and protein synthesis and a decrease of the response of intracellular cholesterol synthesis to changes in the extracellular concentrations of lipoprotein. Various morphological techniques showed virtually no binding and internalization of LDL by keratinocytes cultured at the high-calcium level, whereas both were observed at the low-calcium level. Once internalized, the LDL was delivered to dense bodies representing lysosomes. It is concluded that in human epidermal keratinocytes, the expression of the LDL receptor and the endogenous synthesis of cholesterol are regulated by the conditions determined by the differentiation stage of the cells.     

Differentiation of cultured human keratinocytes: Effect of culture conditions on lipid composition of normal vs. malignant cells

Summary Differentiation in keratinocytes can be experimentally modulated by changing the culture conditions. When cultured under conventional, submerged conditions, the extent of cellular differentiation is reduced in the presence of low calcium medium and is enhanced in medium containing physiologic calcium concentrations. Moreover cultures grown at the air-medium interface or on a dermal substrate, or both, differentiate even further. Herein we report the effect of culture conditions on lipid composition in normal human keratinocytes and three squamous carcinoma cell (SCC) lines that vary in their capacity to differentiate as assessed by cornified envelope formation. Under submerged conditions, the total phospholipid content was lower, triglyceride content higher, and phospholipid: neutral lipid ratio lower in direct correlation to the degree of differentiation in these cultures. When grown at the air-medium interface on the-epidermized dermis, evidence of further morphologic differentiation was found only for well-differentiated SCC cells and normal keratinocytes. Similarly, the phospholipid content remained high in poorly differentiated SCC cells and it, decreased modestly in well-differentiated SCC cells and markedly in normal keratinocytes. In all cell lines the triglyceride content was increased and cholesterol content decreased when compared to parallel submerged cultures, but these differences were most pronounced in well-differentiated cell lines. Acylceramides and acylglucosylceramides were found only in normal keratinocytes and only under the most differentiation-enhancing conditions. These studies demonstrate differentiation-related changes in the lipid content of both normal and neoplastic keratinocytes. This work was supported in part by NATO Scientific Award (RG 8510056).     

Association of EGF and LDL receptors with the cytoskeleton of cultured keratinocytes

In this paper we demonstrate that isolated cytoskeletons of normal keratinocytes cultured under differentiation inducing conditions exhibit a high level of epidermal growth factor (EGF) binding. This binding is approximately 300% higher than the binding of intact cells. In contrast, various squamous carcinoma cell lines or normal keratinocytes cultured under differentiation retarding conditions exhibit EGF binding to isolated cytoskeletons which is around 10-20% of the binding to intact cells. Incubation of normal keratinocytes in the presence of arotinoid ethyl sulfone resulted in a marked decrease of the ability of the cells to differentiate, and a decrease of EGF binding to isolated cytoskeletons. These results suggest a close relationship between the differentiation capacity of the cells and the presence of cytoskeleton-associated EGF receptors. Similar results were obtained for low density lipoprotein (LDL) binding.     

Comparison of the inhibitory effect of polyunsaturated fatty acids on prostaglandin synthesis I oral squamous carcinoma cells

Polyunsaturated fatty acids (PUFAs) have been shown to suppress the growth rate of human osteogenic sarcoma cells and to have selective cytotoxic activity against human cancer cells. The purpose of this study was to investigate the efficacy of various PUFAs on inhibition of prostaglandin (PG) synthesis by oral squamous carcinoma cells (SCC-25). A significant inhibition of PGE2 and PGF2 alpha synthesis in SCC-25 was observed by all PUFAs tested except in the case of linoleic acid (LA) at 10 microM level. At 10 microM level the rank order of inhibition of PG synthesis by PUFAs was docosahexaenoic (DHA) greater than eicosapentaenoic (EPA) + DHA greater than dihomogamma-linolenic (DGLA) greater than EPA greater than alpha-linolenic (ALA) greater than linoleic (LA). At 50, 75, 100 microM the rank order of inhibition was DGLA greater than EPA greater than EPA + DHA greater than DHA greater than ALA greater than LA.     

Lipid content and metabolism of human keratinocyte cultures grown at the air-medium interface

The differentiation of human keratinocytes in most culture systems is incomplete; e.g., lamellar bodies, the characteristic lipid-delivery organelles of epidermis, are not present. Moreover, their lipid profile does not reflect the distinctive composition found in cornifying epidermis. In contrast, keratinocytes that grow at an air-medium interface exhibit more complete differentiation. In this study, we compared the elaboration of lamellar bodies, the lipid content, and the lipid metabolism of human keratinocytes, cultured both under standard immersed conditions and after lifting to an air-medium interface. Whereas submerged cultures neither elaborated lamellar bodies nor displayed a lipid distribution characteristic of cornifying epidermis, lifted cultures displayed advanced cornification, elaborated lamellar bodies which were deposited in intercellular domains, and a lipid profile more typical of cornifying epidermis. Moreover, lipid biosynthesis was 5-10-fold more active in lifted than in immersed cultures, and was not inhibited by exogenous lipoproteins. These findings are consistent with recent studies that demonstrate both high rates of lipogenesis in differentiating layers of the epidermis as well as autonomy of lipogenesis from the influence of circulating lipoproteins. Thus, the lipid content and metabolism of human keratinocyte cultures, grown at an air-medium interface, demonstrate features that simulate the epidermis.     

Influence of low density lipoproteins on vascular smooth muscle cell growth and motility: modulation by extracellular matrix.

Low density lipoproteins (LDL) are thought to play a major role in cardiovascular diseases such as atherosclerosis. Much remains to be done to understand the cellular effects of LDL and how the extracellular matrix (ECM) influences these effects. We found that LDL produced a dose dependent increase in vascular smooth muscle cell (SMC) proliferation. The ECM altered the proliferative response of SMC to LDL: on collagen I there was a 66% inhibition, endothelial cell derived-ECM a 2-fold increase, and collagen IV no difference in proliferation compared to paired controls. LDL affected SMC motility (cell area and shape factor) but the extent and direction of the effect depended on whether the cells were cultured on uncoated or coated dishes. LDL treated cultures had a 5-fold lower migration rate but net movement was not different, suggesting that LDL decreased SMC random movement. There was a dose-dependent accumulation of lipid by SMC incubated with LDL and, subsequently, cytoplasmic lipid droplets were observed. Cells cultured on uncoated plates showed an increased cholesterol content as a function of LDL concentration. In contrast, cells cultured on a collagen IV matrix showed no net change in cholesterol content over the range of LDL concentrations studied. Hence, the uptake of LDL cholesterol appears to be completely inhibited by this matrix. These studies indicate that the influence of LDL on several SMC parameters is modulated by ECM components.     

Lipid, apolipoprotein, and lipoprotein synthesis and secretion during cellular differentiation in caco-2 cells

Summary Although Caco-2 cells are frequently employed for the study of enterocyte lipid metabolism, variable results have been reported regarding their ability to synthesize and secrete lipids and apolipoproteins. The major goal of this investigation is to examine the capacity of Caco-2 cells to elaborate and secrete lipids, lipoproteins, and apolipoproteins at different degrees of morphological and functional differentiation. Cells were cultured in medium with 5% fetal bovine serum (FBS), on permeable polycarbonate filters from 2 to 30 d in the presence of ¹⁴C-oleate or ³⁵S-methionine. Cellular differentiation, as assessed by morphology (light and electron microscopy), transepithelial resistance, free fatty acid flux, and sucrase activity, progressed steadily up to 20 d of culture. Caco-2 cells esterified oleic acid mainly into phospholipids, triglycerides (TG), and smaller amounts of cholesterol esters. Lipid synthesis began as early as 2 d, and TG secretion was enhanced with increased duration of culture. However, very low efficiency of lipid export was observed at all levels of differentiation, reaching a maximum of only 6% of intracellular lipids. VLDL and LDL were the dominant lipoproteins secreted, with HDL comprising <20% of the total. VLDL secretion increased, while LDL decreased, whereas the lipid composition of lipoproteins varied little with increasing duration of culture. Apoprotein B and A-I synthesis and secretion increased markedly from 11 to 20 d of culture. The ratio of apo B-100/B-48 decreased between 11 and 30 d, consistent with enhanced apo B editing of more mature enterocytes. Taken together, our data suggest that from 20 d of culture, Caco-2 cells are morphologically and functionally mature, capable of lipid esterification, and lipoprotein and apolipoprotein synthesis. However, despite their functional and morphological similarities to mature enterocytes, Caco-2 cells have a very limited lipid export capacity.     

Free sterol metabolism and low density lipoprotein receptor expression as differentiation markers of cultured human keratinocytes

In contrast to most tissues, epidermis and its derivatives appear to lack low density lipoprotein (LDL) receptors and exhibit sterologenesis rates unaffected by circulating lipoprotein (LP) cholesterol content. Since LDL receptors have been demonstrated in both cultured squamous cell carcinoma cells and human foreskin keratinocytes, when maintained in low-calcium media, LDL receptor expression may be related to keratinocyte differentiation. We compared receptor binding and internalization of LDL-gold in normal keratinocytes at different stages of growth at physiological calcium concentrations (early, 3-5 days; preconfluent, 6-10 days; postconfluent, 12-17 days), and correlated receptor expression with sterologenesis in LP-replete vs.-depleted media. Whereas in early cultures about 60% of sterologenesis was LP dependent, this fraction declined in preconfluent and confluent cultures despite continued culture growth and little decline in total sterologenesis. Accordingly, LDL receptors were most evident in early cultures, declining in preconfluent cultures in parallel with the decrease in LP-dependent sterol synthesis. In contrast, sterologenesis in human foreskin fibroblasts was profoundly influenced by exogenous LP at all stages of confluence; total and LP-dependent sterologenesis declined in parallel with growth cessation. These studies represent the first demonstration that normal keratinocytes express functional LDL receptors at physiologic calcium concentrations. Moreover, they demonstrate that LDL receptor expression in keratinocytes, in contrast to fibroblasts, can only in part be attributed to growth requirements. Instead, loss of LDL receptor expression serves as a distinctive marker of keratinocyte differentiation and may reflect the specific functional requirements of the epidermis in vivo.     

Comparison of the inhibitory effect of polyunsaturated fatty acids on prostaglandin synthesis. II. Fibroblasts

In a previous publication we reported that PUFAs of the n-6 and n-3 series caused significant inhibition of synthesis of both PGE2 (28.4-92.8%) and PGF2 alpha (24.4-84.0%) in the oral squamous carcinoma cell line SCC-25. In this report we describe the inhibitory effect of the same acids on PG synthesis in normal human gingival fibroblasts under the same experimental conditions. It was found that a combination of EPA + DCHA (6:4), DCHA and ALA caused significant reduction in synthesis of PGE2 (10.1-87.8%) and PGF2 alpha (14.0-54.6%) at the four dose levels studied. The rank order of potency of acids in reduction of PG synthesis was: EPA + DCHA greater than DCHA greater than EPA greater than ALA greater than LA greater than DGLA greater than GLA. The data suggest that although PUFAs are effective inhibitors of PG synthesis by gingival fibroblasts and SCC-25, the fibroblast is less susceptible to the inhibitory effect of fatty acids.     

ANO1对口腔鳞癌SCC-25细胞株的影响

采用免疫组化检测160例口腔鳞癌组织及相应正常组织的ANO1表达,并进行多项体外买验,以明确ANO1对SCC-25细胞迁移的影响。结果显示,正常组织中的ANO1阳性表达明显低于口腔鳞癌组织;有淋巴结转移的口腔鳞癌组织的ANO1阳性表达显著高于无转移的口腔鳞癌组织;多项体外实验表明,ANO1过表达有利于SCC-25细胞的迁移;尼氟灭酸能减缓细胞的迁移速度。综上所述,ANO1促进了口腔鳞癌患者体内的肿瘤转移,并增加了SCC-25细胞的体外移动、侵袭、伸展、剥脱能力,有望成为治疗口腔鳞癌的新的靶点。     

Oxidized LDL bind to nonproteoglycan components of smooth muscle extracellular matrices

Arterial wall lipid retention is believed to be due primarily to ionic interactions between lipoproteins and proteoglycans. Thus, oxidized low density lipoproteins (LDL), with decreased positive charge relative to native LDL, should have decreased interaction with negatively charged proteoglycans. However, oxidized LDL does accumulate within arterial lesions. Therefore, this study investigated the binding of native and oxidized LDL to a complex smooth muscle extracellular matrix and the role of ionic charge interactions in their binding. LDL was modified with 2,2-azo-bis(2-amidinopropane)-2HCl, hypochlorite, soybean lipoxygenase, and phospholipase or copper sulfate. The extracellular matrix had 15- to 45-fold greater binding capacity for the different forms of oxidized LDL than for native LDL. However, the affinity of binding for all forms of oxidized LDL was high (K(a) = approximately 10(-9) M) and was similar to that for native LDL. Preincubation of the lipoproteins with chondroitin sulfate decreased the binding of native LDL, but had no effect on the binding of oxidized LDL. Digestion of matrices with chondroitin ABC lyase and heparinase decreased the binding of native LDL, but increased the binding of oxidized LDL; matrix digestion with pronase or trypsin markedly reduced the binding of both native and oxidized LDL.Thus, the binding of native LDL involves matrix proteoglycans, whereas the binding of oxidized LDL involves a nonproteoglycan component(s) of the matrix. The markedly enhanced retention of oxidized LDL compared with native LDL may play an important role in the progression of atherosclerosis.     

Scavenger receptor BI transfers major lipoprotein-associated phospholipids into the cells

The phospholipids of lipoproteins can be transferred to cells by an endocytosis-independent uptake pathway. We analyzed the role of scavenger receptor BI (SR-BI) for the selective cellular phospholipid import. Human monocytes rapidly acquired the pyrene (py)-labeled phospholipids sphingomyelin (SM), phosphatidylcholine, and phosphatidylethanolamine from different donors (low and high density lipoproteins (LDL, HDL), lipid vesicles). The anti-SR-BI antibody directed against the extracellular loop of the membrane protein lowered the cellular import of the phospholipids by 40-80%. The phospholipid transfer from the lipid vesicles into the monocytes was suppressed by LDL, HDL, and apoprotein AI. Transfection of BHK cells with the cDNA for human SR-BI enhanced the cellular import of the vesicle-derived py-phospholipids by 5-6-fold. In the case of the LDL donors, transfer of py-SM to the transfected cells was stimulated to a greater extent than the uptake of the other py-phospholipids. Similar differences were not observed when the vesicles and HDL were used as phospholipid donors. The concentration of LDL required for the half-maximal phospholipid import was close to the previously reported apparent dissociation constant for LDL binding to SR-BI. The low activation energy of the SR-BI-mediated py-phospholipid import indicated that the transfer occurs entirely in a hydrophobic environment. Disruption of cell membrane caveolae by cyclodextrin treatment reduced the SR-BI-catalyzed incorporation of py-SM, suggesting that intact caveolae are necessary for the phospholipid uptake. In conclusion, SR-BI mediates the selective import of the major lipoprotein-associated phospholipids into the cells, the transfer efficiency being dependent on the structure of the donor lipoprotein.     

The influence of oxidatively modified low density lipoproteins on expression of platelet-derived growth factor by human monocyte-derived macrophages

Platelet-derived growth factor (PDGF) is secreted by several cells that participate in the process of atherogenesis, including arterial wall monocyte-derived macrophages. Macrophages in human and non-human primate lesions have recently been demonstrated to contain PDGF-B chain protein in situ. In developing lesions of atherosclerosis, macrophages take up and metabolize modified lipoproteins, leading to lipid accumulation and foam cell formation. Oxidatively modified low density lipoproteins (LDL) have been implicated in atherogenesis and have been demonstrated in atherosclerotic lesions. The effects of the uptake of various forms of modified LDL on PDGF gene expression, synthesis, and secretion in adherent cultures of human blood monocyte-derived macrophages were examined. LDL oxidized in a cell-free system in the presence of air and copper inhibited the constitutive expression of PDGF-B mRNA and secretion of PDGF in a dose-dependent fashion. Oxidatively modified LDL also attenuated lipopolysaccharide-induced PDGF-B mRNA expression. These changes were unrelated to the mechanism of lipid uptake and the degree of lipid loading and were detectable within 2 h of exposure to oxidized LDL. The degree of inhibition of both basal and lipopolysaccharide-induced PDGF-B-chain expression increased with the extent of LDL oxidation. Monocyte-derived macrophages exposed to acetylated LDL or LDL aggregates accumulated more cholesterol than cells treated with oxidized LDL, but PDGF expression was not consistently altered. Thus, uptake of a product or products of LDL oxidation modulates the expression and secretion of one of the principal macrophage-derived growth factors, PDGF. This modulation may influence chemotaxis and mitogenesis of smooth muscle cells locally in the artery wall during atherogenesis.     

Cancer cells responsible for humoral hypercalcemia express mRNA encoding a secreted form of ODF/TRANCE that induces osteoclast formation

A novel cDNA encoding a secreted form of osteoclast differentiation factor/tumor necrosis factor-related activation-induced cytokine (sODF/TRANCE, GenBank Accession No. AB037599) was sequenced from 5' RACE cDNA clones of squamous cell carcinoma cell lines, SCC-4 and T3M-1 Cl.2, of which parental malignant tissues had caused severe humoral hypercalcemia. The sODF/TRANCE cDNA was composed of unknown 5' end sequence followed by the 100% identical sequence of the ODF/TRANCE extracellular domain-coding region. The longest open reading frame (ORF) of the novel cDNA completely matched the 3' end of the ORF of the ODF/TRANCE cDNA encoding C-terminal amino acid residues (74-318) in the extracellular region. The corresponding protein that reacted with the antibody specific for the extracellular domain of ODF/TRANCE was detected in the culture media conditioned by the cancer cells. Furthermore, human promyeloblastic leukemia cells, HL60, differentiated into osteoclast-like cells (OCLs) when cultured in the media conditioned by SCC-4 and T3M-1 Cl. 2 cells. The differentiation of HL60 cells into OCLs was inhibited by the anti-ODF/TRANCE antibody. These results strongly suggest that sODF/TRANCE plays an important role in enhanced bone-resorption in humoral hypercalcemia of malignancy.