Proc. Natl. Acad. Sci. USA Vol. 74, No. 8, pp. 3429-32, August 1977
Cell Biology
Fibroblast heterogeneity and prostaglandin regulation of subpopulations (DNA synthesis/mitosis/connective tissue/collagen/gingiva)
S. DAVID Ko, RoY C. PAGE, AND A. S. NARAYANAN Departments of Pathology and Periodontics and The Center for Research in Oral Biology, University of Washington, Seattle, Washington 98195
Communicated by Earl P. Benditt, May 20, 1977
The effects of prostaglandin E2 (PGE2) upon the synthesis of protein and DNA, and membrane transport of proline and thymidine, by human diploid fibroblasts were studied. At a concentration range of 1-10 pM, PGE2 inhibited protein synthesis and membrane transport by 45-50%. Serumactivated DNA synthesis and thymidine transport were also inhibited by approximately 50% in cells made quiescent and synchronous by serum deprivation. To determine whether prostaglandin inhibits some of the cells completely or all of the cells partially, radioautographic and cell-counting experiments were done. In cultures pulse-labeled with [3Hjthymidine 12-33 hr after serum activation, prostaglandin exposure reduced the number of labeled nuclei by 42%. Sixty-five hours after serum activation, the total cell numbers present in the PGE2-exposed cultures were reduced by 25%. Furthermore, in the fibroblast cultures derived from cells previously maintained in 10 pM PGE2 for 14 days, PGE2 had no effect on DNA synthesis, indicating that the PGE2-sensitive cells had disappeared from the cultures. Thus, PGE2 appears to inhibit growth and synthesis of a subpopulation of cells while not affecting the remaining insensitive cells. Prostaglandins may play an important role in connective-tissue differentiation an in some pathologic alterations by regulating fibroblast subpopulations. ABSTRACT
The prostaglandins (PGs) are a family of 20-carbon unsaturated fatty acids derived from membrane phospholipid precursors through the action of a membrane-bound enzyme referred to as prostaglandin synthetase (1). These substances, which are generally classified as local or cellular hormones, are found in normal and inflamed tissues (2, 3) and in various inflammatory exudates (2-4). They have been implicated in a broad range of biologic phenomena including acute and chronic inflafmmation (2-5), normal and pathologic immune reactions (6-9), connective-tissue alterations and fibrosis (10-15), and pathologic bone resorption (16, 17). The effects of the prostaglandins upon connective tissues remain ill defined (10, 12-15, 18-20). We have studied-the effects of prostaglandin upon the growth and synthetic activities of diploid fibroblasts derived from normal human connective tissue. Our data show that only a subpopulation of approximately half the cells activated to synthesize DNA by serum exposure respond to PGE2, while the remaining cells are unaffected. In the responding subpopulation, membrane transport and synthetic activity appear to be inhibited completely, resulting, with the passage of time, in the overgrowth of the nonresponding cell population. EXPERIMENTAL PROCEDURES Fibroblasts were derived from an explant of normal human gingiva as described by Narayanan and Page (21). The morphologic characteristics (22) and synthetic activities (21) of the The costs of publication of this article were defrayed in part by the payment of page charges from funds made available to support the research which is the subject of the article. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
3429
cell line used (HGF4) have been described; the cells are diploid and behave in vitro in a manner similar to skin fibroblasts. Total synthetic activity and amino acid transport were evaluated by measuring the uptake of radiolabeled amino acids by confluent cultures maintained in 60-mm Falcon petri dishes in Dulbecco-Vogt medium with 10%o fetal calf serum (FCS) and sodium ascorbate at 50 Mg/ml as described by Hassell et al. (23). DNA content was measured by the method of Kissane and Robins (24). Membrane transport of proline was evaluated in cultures prepared in a similar manner in 35-mm Falcon petri dishes (25). For measuring thymidine transport and DNA synthesis, cells were made quiescent and synchronous by seeding 0.75 X 106 cells of transfer number 6 to 10 into 75-cm2 Falcon flasks in 10 ml of medium as described above, and incubating for 5 days with one change of medium, followed by incubation for 24-48 hr in complete medium without serum. The cells were then harvested by exposure to 0.05% trypsin, washed in medium with 10% FCS, and placed in RPMI 1640 medium without serum. Exposure of the cells to trypsin and to serum was less than 3 min each. The harvested cells were seeded into microtest wells or petri dishes or onto cover slips. In cultures prepared under these conditions, less than 1.0% of the cell nuclei become labeled during a subsequent 24-hr pulse with [3H]-
thymidine. RESULTS PGE2 inhibits protein synthetic activity by human diploid fibroblasts, as measured by the incorporation of labeled amino acids into nondialyzable material. Maximum inhibition of about 50% occurred at a PG concentration range of 1-10MuM (Fig. 1). As illustrated in Table 1, DNA synthesis by synchronous, quiescent cells, activated by serum exposure, was inhibited 45% by PGE2 at 10 MM. Because the inhibition observed could have resulted from either a quantitative reduction in label incorporated without alteration of the time course of DNA synthesis or from a delay in synthesis, the time course of DNA synthesis was also evaluated, using the same experimental system. Cells made quiescent and synchronous and subsequently exposed to 10% FCS serum begin DNA synthesis after a lag period of approximately 12-18 hr. During this lag period, DNA precursors are not transported into the cells. In control cultures not containing prostaglandin, label appeared in DNA by 18 hr, became maximal at 24 hr, then rapidly subsided by 33 hr (Fig. 2). The presence of PGE2 did not alter the time course of DNA synthesis but, as noted in the previous experiment, reduced the magnitude. DNA synthesis did not occur in cultures containing prostaglandin but no serum; thus, PGE2 is not mitogenic for
these fibroblasts. The idea that prostaglandins may inhibit protein and DNA synthesis by interfering with the transport of precursor subAbbreviations: PG, prostaglandin; FCS, fetal calf serum.
3430
Cell Biology: Ko et al.
Cell Biology
Fibroblast heterogeneity and prostaglandin regulation of subpopulations (DNA synthesis/mitosis/connective tissue/collagen/gingiva)
S. DAVID Ko, RoY C. PAGE, AND A. S. NARAYANAN Departments of Pathology and Periodontics and The Center for Research in Oral Biology, University of Washington, Seattle, Washington 98195
Communicated by Earl P. Benditt, May 20, 1977
The effects of prostaglandin E2 (PGE2) upon the synthesis of protein and DNA, and membrane transport of proline and thymidine, by human diploid fibroblasts were studied. At a concentration range of 1-10 pM, PGE2 inhibited protein synthesis and membrane transport by 45-50%. Serumactivated DNA synthesis and thymidine transport were also inhibited by approximately 50% in cells made quiescent and synchronous by serum deprivation. To determine whether prostaglandin inhibits some of the cells completely or all of the cells partially, radioautographic and cell-counting experiments were done. In cultures pulse-labeled with [3Hjthymidine 12-33 hr after serum activation, prostaglandin exposure reduced the number of labeled nuclei by 42%. Sixty-five hours after serum activation, the total cell numbers present in the PGE2-exposed cultures were reduced by 25%. Furthermore, in the fibroblast cultures derived from cells previously maintained in 10 pM PGE2 for 14 days, PGE2 had no effect on DNA synthesis, indicating that the PGE2-sensitive cells had disappeared from the cultures. Thus, PGE2 appears to inhibit growth and synthesis of a subpopulation of cells while not affecting the remaining insensitive cells. Prostaglandins may play an important role in connective-tissue differentiation an in some pathologic alterations by regulating fibroblast subpopulations. ABSTRACT
The prostaglandins (PGs) are a family of 20-carbon unsaturated fatty acids derived from membrane phospholipid precursors through the action of a membrane-bound enzyme referred to as prostaglandin synthetase (1). These substances, which are generally classified as local or cellular hormones, are found in normal and inflamed tissues (2, 3) and in various inflammatory exudates (2-4). They have been implicated in a broad range of biologic phenomena including acute and chronic inflafmmation (2-5), normal and pathologic immune reactions (6-9), connective-tissue alterations and fibrosis (10-15), and pathologic bone resorption (16, 17). The effects of the prostaglandins upon connective tissues remain ill defined (10, 12-15, 18-20). We have studied-the effects of prostaglandin upon the growth and synthetic activities of diploid fibroblasts derived from normal human connective tissue. Our data show that only a subpopulation of approximately half the cells activated to synthesize DNA by serum exposure respond to PGE2, while the remaining cells are unaffected. In the responding subpopulation, membrane transport and synthetic activity appear to be inhibited completely, resulting, with the passage of time, in the overgrowth of the nonresponding cell population. EXPERIMENTAL PROCEDURES Fibroblasts were derived from an explant of normal human gingiva as described by Narayanan and Page (21). The morphologic characteristics (22) and synthetic activities (21) of the The costs of publication of this article were defrayed in part by the payment of page charges from funds made available to support the research which is the subject of the article. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
3429
cell line used (HGF4) have been described; the cells are diploid and behave in vitro in a manner similar to skin fibroblasts. Total synthetic activity and amino acid transport were evaluated by measuring the uptake of radiolabeled amino acids by confluent cultures maintained in 60-mm Falcon petri dishes in Dulbecco-Vogt medium with 10%o fetal calf serum (FCS) and sodium ascorbate at 50 Mg/ml as described by Hassell et al. (23). DNA content was measured by the method of Kissane and Robins (24). Membrane transport of proline was evaluated in cultures prepared in a similar manner in 35-mm Falcon petri dishes (25). For measuring thymidine transport and DNA synthesis, cells were made quiescent and synchronous by seeding 0.75 X 106 cells of transfer number 6 to 10 into 75-cm2 Falcon flasks in 10 ml of medium as described above, and incubating for 5 days with one change of medium, followed by incubation for 24-48 hr in complete medium without serum. The cells were then harvested by exposure to 0.05% trypsin, washed in medium with 10% FCS, and placed in RPMI 1640 medium without serum. Exposure of the cells to trypsin and to serum was less than 3 min each. The harvested cells were seeded into microtest wells or petri dishes or onto cover slips. In cultures prepared under these conditions, less than 1.0% of the cell nuclei become labeled during a subsequent 24-hr pulse with [3H]-
thymidine. RESULTS PGE2 inhibits protein synthetic activity by human diploid fibroblasts, as measured by the incorporation of labeled amino acids into nondialyzable material. Maximum inhibition of about 50% occurred at a PG concentration range of 1-10MuM (Fig. 1). As illustrated in Table 1, DNA synthesis by synchronous, quiescent cells, activated by serum exposure, was inhibited 45% by PGE2 at 10 MM. Because the inhibition observed could have resulted from either a quantitative reduction in label incorporated without alteration of the time course of DNA synthesis or from a delay in synthesis, the time course of DNA synthesis was also evaluated, using the same experimental system. Cells made quiescent and synchronous and subsequently exposed to 10% FCS serum begin DNA synthesis after a lag period of approximately 12-18 hr. During this lag period, DNA precursors are not transported into the cells. In control cultures not containing prostaglandin, label appeared in DNA by 18 hr, became maximal at 24 hr, then rapidly subsided by 33 hr (Fig. 2). The presence of PGE2 did not alter the time course of DNA synthesis but, as noted in the previous experiment, reduced the magnitude. DNA synthesis did not occur in cultures containing prostaglandin but no serum; thus, PGE2 is not mitogenic for
these fibroblasts. The idea that prostaglandins may inhibit protein and DNA synthesis by interfering with the transport of precursor subAbbreviations: PG, prostaglandin; FCS, fetal calf serum.
3430
Cell Biology: Ko et al.
