- Email: [email protected]
Transforming growth factor-β1 expression during placental development
Transforming growth factor-~l expression during placental development Lauren J. Dungy, MD: Tariq A. Siddiqi, MD: and Sohaib Khan, PhD b Cincinnati, Ohio Placental growth has several malignant characteristics, including properties of invasiveness, rapid cell proliferation, and a lack of cell contact inhibition. These malignant characteristics of placental development are strictly regulated throughout normal gestation, because placental growth is limited in both extent and duration. Transforming growth factor-I3, inhibits growth of many normal and malignant cell lines. In this study, using Northern blot analysis, we found transforming growth factor-I3, expression to occur in human placenta throughout gestation. Peak expression was noted at midgestation (near 17 weeks) and again in late gestation (near 34 weeks). Immunohistochemical analysis localized transforming growth factor-I3, protein expression to the syncytiotrophoblastic layer. The process of trophoblastic invasion of the decidua and myometrium is usually complete by 18 weeks of gestation, and absolute growth of the placenta ceases in late gestation (near 35 weeks). The time frames of maximal transforming growth factor-I3, expression noted in our studies correlate with these events. We speculate that peak transforming growth factor-I3, expression at these stages of placental development is suggestive of its regulation of both trophoblastic invasion and proliferation. (AM J OSSTET GVNECOL 1991 ;165:853-7.)
Key words: Transforming growth factor-I3., human placenta
Placental implantation and subsequent growth and development have been described asa pseudomalignant process. Similarities between trophoblastic cell growth and malignant cells include invasiveness, rapid cell proliferation, lack of cell contact inhibition, and a degree of immune privilege. These malignant characteristics of placental development appear to be strictly regulated throughout normal gestation, because placental growth is limited in both its extent and duration. In fact, during embryogenesis and placentation tissues and organs develop in a sequential pattern, implying that genes coding for particular proteins are turned on and off in a precise and well-regulated manner. It is now becoming apparent that growth inhibitors play an important role in maintaining tissue homeostasis. Peptide growth factors, active in common regulatory pathways, may be important regulators of placental develo~ment.
-Transforming growth factor-l3. (TGF-I3.) is a polyFrom the Division of Maternal-Fetal Medicine, Departments of Obstetrics and Gynecology,' and the Department of Anatomy and Cell Biology,' University of Cincinnati College of Medicine. Supported in part by a Research Grant to the Department of Obstetrics and Gynecology, University of Cincinnati College of Medicine, Cincinnati, Ohio. Presented at the Eleventh Annual Meeting of the Society of Perinatal Obstetricians, San Francisco, California, January 28-February 2,
1991. Reprint requests: Lauren]. Dungy, MD, Division of Maternal-Fetal Medicine, Department of Obstetrics and Gynecology, University of Cincinnati College of Medicine, 231 Bethesda Ave., Cincinnati, OH 45267-0526.
6/6/30897
peptide originally identified in tumor cells and postulated to be involved in transformation and neoplastic growth. Its presence in numerous tissues,I-4 however, implies a normal physiologic role that appears to be multifunctional. TGF-131 has been shown to stimulate growth of some cell types but to inhibit growth of others, to stimulate growth of fibroblasts and promote wound healing," 6 and to induce differentiation of leukemic and bronchial epithelial cell lines. 7• S A fundamental property of TGF-131 is its ability to inhibit proliferation of both normal and malignant cell lines. TGF13. has been shown to inhibit growth of different ovarian cancer cell lines, as well as some human leukemic cell lines. s . g As such, we hypothesized that TGF-I3. may play an important role in the inhibition of trophoblastic invasion of the myometrium during placental development. In humans, this process is largely limited to the eighth week through the eighteenth week of gestation. 10 We evaluated the temporal expression of TGF-131 in human placenta to begin to examine this hypothesis.
Material and methods Collection of placental tissue. Placental tissue was obtained after curettage, spontaneous delivery, or cesarean section and immediately placed in liquid nitrogen. Informed consent was obtained from patients in all instances, in accordance with the protocol approved by our institutional review board. Time frames of placental tissue studied were chosen to correlate with early, mid, and late gestation. Placental tissue obtained from a total of 20 patients was used for analysis. Initially, a
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WEEKS GESTATION Fig. 1. Graph showing results of scanning densitometry of blots of five representative RNA samples. TGF-~ , expression is noted throughout gestational period shown , with peak expression at 17 and 34 weeks' gestation.
panel of total ribonucleic acid (RNA) from placental tissues ranging from 6 to 40 weeks' gestation was studied with Northern blot analysis. These studies were repeated with total RNA from different placental tissues of similar gestational ages . Three panels of RNA were studied. Additionally, r\orthern blot analysis was performed with 100 f.Lg of ' <" .J RNA from placental tissue of IS through 19 weeks' gestational age and 32 through 34 weeks' gestational age to furt her evaluate time frames where peak ex :) 1 ession had previously been identified. RNA isolation. Total RNA was isolated from the placental tissues according to the technique of Chomzynski and Sacchi." Briefly, approximately 2 gm of placental tissue was homogenized in 10 ml extractio n solution (4 mol/L guanidine thiocyanate ; 25 mmol / L sodium citrate, pH 7.0; 0 5. % sarcosyl; 0.1 mol/L 2-j)-mercaptoethanol ; 2 mol/L sodium acetate; and 10 ml phenol). The homogenate was mixed with chloroform / isoamyl alcohol (2 ml of 49: 1 solution) and chilled on ice for 15 minutes. After centrifugation for 20 minutes at 10,000 g at 4° C, the aqueous phase was removed and precipitated with one volume of isopropanol at - 20° C for 2 hours. This was fo llowed by a second centrifugation for 20 minutes at 10,000 g at 4° C. The resultant pellet was dissolved in extraction solution (600 f.LI) and precipitated with isopropanol (I : I,vol/ vol) and sodium acetate (I: 10, vol/vol) at -20° C for approximately 2 hours. After centrifugation for 10 minutes at 4° C, the pellet was washed with 70 % ethanol and vacuum dried for approximately 5 minutes. The pellet was then resuspended in diethylprocarbonate-treated water. Total RNA concentration was quantitated by absorption spectrophotometry at 260 nm. Northern blot analysis. RNA samples (100 f.Lg) were
resolved by electrophoresis on formaldehyde agarose gels and transferred to nitrocellulose membranes by towel blotting. The integrity and quantity of each RNA sample were ensured by stainin g the ge l with ethidium bromide and visualizing the gel under ultraviolet transillumination. The filters were prehybridized in hybridization solution containing 10 X Denhardt's solution , 0.1 % sod ium dodedecyl sulfate, 100 f.Lg / ml single standard sperm deoxyribonucleic acid, 50% deionized forma mide, and a 5 x solution of 3 mollL sodium chloride, 0.1 mol/L sodium phosphate, 0.02 mol/L ethylenediaminetetraacetic acid, and sodium hydroxide and then hybridized in the same solution containing a phosphorus 32-labeled human TGF-j), complementary deoxyribonucleic acid probe ( I x 10'; counts / min / ml) at 42° C overnight. After four washings (final wash I X saline sodium citrate buffer with 0.1 % sodium dodedecyl sulfate at 55° C for 20 minutes) the filters were subjected to autoradiography. The position of 18S and 28S RNA was determined, and the single band observed, which corresponded to the position of TGF-f3, messenger RNA (mRNA) (2.1 kb), was quantitated by scanning densitometry with a Hoefer GS 300 (Hoefer Scientific Instruments, San Francisco) gel scanner. Our initial studies with 25 and 50 f.Lg of total RNA revealed expression only at 17 and 34 wee ks' gestation . In later blots TGF-f3, expression was noted at all time points when 100 f.Lg of total RNA was used. For this reason 100 f.Lg samples were used in all experiments reported in this study. Immunohistochemical analysis. Placental sections (10 f.Lm) from 17 and 34 weeks' gestation were fixed in chi lled methanol, followed by fixation in sodium periodate, lysine, and paraformaldehyde solution. The sections were then incubated at room temperature with a phosphate-buffered saline solution containing bovine serum albumin (1%) and 0.1 % Triton-X-lOO (Fischn Scientific, Fair Lawn , N.J .) compound for 1O minutes, followed by a 30-minute incubation with phosphatebuffered saline solution containing 0.2% gelatin to minimize background contamination. The sections were then incubated with polyclonal rabbit antibody against human TGF-f3! (1: 50 dilution in phosphate-buffered saline solution-bovine serum albumin) for I hour at 37° C. TGF-f3, antibody used in this study was obtained from Collage n Corporation, Palo Alto, Calif. After three washings with phosphate-buffered saline solution containing 0.2% gelatin fO!' 15 minutes each, the specimens were incubated with fluorescein-conjugated goat anti-rabbit polyclonal antibody (I : 50 dilution in phosphate-buffered saline solution -bovine serum albumin) for 45 minutes at 37° C. After three washings in phosphate-buffered saline solution containing 0.2% gelatin, immunofluore·scent sites were visualized in an epiflu-
Voillme 16!\ NlImber 4. Pal'! I
Transforming growth factor-I3, expression in human placenta
855
Fig. 2. Northern blot of total RNA samples isolated from placental tissues near midgestation. TGF13, expression is noted throughout (15 to 19 weeks). Maximal expression was found at 17 weeks' gestation by densitometric quantitation.
orescence microscope (Leitz, Wetzler, Germany) . Sequential sections were treated in a similar manner with nonimmune rabbit serum substituted at the same concentration for TGF-f3, antibody to serve as control. Results
With Northern blot analysis and 100 fJ..g of total RNA, we were able to identify TGF-f3, mRNA expression throughout the gestational ages studied (6 through 40 weeks). Fig. 1 represents the continuous scanning densitometry graph of a blot of equal amounts of total RNA isolated from placental tissue ranging from 9 to :H weeks' gestation. Expression of TGF-f3, was noted throughout the time points examined, with peaks of expl'ession noted at 17 and 34 weeks' gestation. This is consistent with our initial findings of TGF-f3, mRNA expression only at 17 and 34 weeks' gestation using lower amounts of total RNA (25 and 50 fJ..g). Northern blot analysis of total RNA isolated from tissue samples ranging from 15 to 19 weeks' gestation revealed TGF-f3, expression throughout these time points (Fig. 2). Maximal expression was observed at 17 weeks by densitometric quantitation. Analysis of total RNA isolated from tissue of 32 to 34 weeks' gestation revealed minimal expression in 32 and 33 week samples with a striking increase of expression occurring at 34 weeks (Fig. 3). We have looked at the expression of c-jun and jun-B in human placenta. These protooncogenes have been associated with cellular growth processes, including both proliferation and differentiation. Their patterns of expression did not correspond to that found with TGF-f3, (unpublished data). This would imply that the
Fig. 3. Northern blot of LOtal RNA samples of placental tissue in late gestation. Minimal expression occurred at 32 and 33 weeks with increased expression at 34 weeks.
increased expression of TGF-f3, near 17 and 34 weeks' gestation is not related to a general phenomenon. After identification of peak TGF-f3, mRNA expression in 17 and 34 weeks' gestational age placental tissue, we used immunohistochemical analysis to localize TGFf3, protein expressinn in these tissues. TGF-f3, protein expression was localized to the periphery of placental villi corresponding with the syncytiotrophoblastic layer (Fig. 4). There was an absence of staining of cell types of the villous core. Control specimens did not show this pattern of staining.
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Dungy, Siddiqi, and Khan
October 1991 Am J Obstet Gynecol
Fig. 4. Immunohistochemical analysis of 17-week placental tissue. TGF-I3, protein expression is localized to syncytiotrophoblastic layer only. (Original magnification x 100.)
Comment The placenta is a very dynamic organ, passing through stages of graft acceptance, cell differentiation, invasive growth, and cessation of both growth and invasiveness. Knowledge about regulatory factors involved in these processes is limited. TGF-J3, promotes proliferation of some cell types but inhibits proliferation of others, depending on cell conditions and concentration. A fundamental property is its ability to inhibit proliferation of both normal and malignant cell lines. The observations made in our study suggest that TGF-J3, is expressed in human placenta throughout gestation. Additionally, two peaks of expression were noted: an initial peak occurring near midgestation at 17 weeks and a second peak in later gestation, near 34 weeks. In humans trophoblastic invasion of the myometrium is largely limited to the eighth through eighteenth weeks of gestation. Large numbers of cytotrophoblastic cells invade the decidua basalis and underlying myometrium. Fusion of cytotrophoblastic cells results in formation of syncytial cells. An absence of mitotic activity is noted in the syncytiotrophoblast.'2 Increased mRNA expression of TGF-J3, near 17 weeks' gestation suggests that it may playa role in inhibition of myometrial trophoblastic invasion. Also, TGF-J3, protein expression was localized to the syncytiotrophoblastic layer with immunohistochemical analysis.
The expression ofTGF-J3" a factor known to be related to inhibition of cell proliferation, in these mitotic endstage cells supports its possible role in the inhibition of cytotrophoblastic myometrial invasion. It is possible that different trophoblastic cell types may express TGFJ3, near midgestation and be involved in myometrial invasion. Our studies, however, were able to identify only TGF-J3, protein expression localized to the villous syncytiotrophoblast with an absence of staining of other cell types. Although we cannot rule out decidual contamination as a source ofTGF-J3, mRNA expression in our specimens, immunohistochemical analysis identified TGF-J3, protein expression only in the syncytiotrophoblastic layer. Absolute growth of human placenta as determined by deoxyribonucleic acid content declines in late gestation beginning at 35 to 36 weeks' gestation, at which time cytotrophoblastic proliferation essentially ceases. 13 TGF-J3, has been shown to inhibit rat trophoblastic cell deoxyribonucleic acid synthesis in a dose-dependent manner.'1 A second peak of TGF-J3, expression near 34 weeks' gestation may therefore be related to this cessation of cytotrophoblastic cell proliferation and decline in placental growth. This was again supported by localization of TGF-I3, protein expression in the syncytiotrophoblastic layer with immunohistochemical analysis. In summary, with Northern blot analysis TGF-I3,
Volume 165 Number 4, Part 1
expression was noted throughout gestation in our study. Peak expression was noted at 17 and 34 weeks' gestation. Immunohistochemical staining revealed TGF-I3, expression restricted to the syncytiotrophoblastic layer. TGF-I3, expression in the syncytiotrophoblast at these time points may be related to inhibition of cytotrophoblastic proliferation occurring at these stages of placental development. Pathologic conditions of trophoblastic in vas ion exhibited in gestational trophoblastic disease and choriocarcinoma represent a deviation from normal trophoblastic growth. We have not examined such tissues. Examination of these tissues with comparison to findings in nonpathologic conditions would be of value in further elucidation of the role of TGF-I3, in regulating trophoblastic myometrial invasion. REFERENCES 1. Sporn M, Roberts A. The transforming growth factorbetas: past, present and future. Ann NY Acad Sci 1990;593: 1-6. 2. Miller D, Pelton R, Deryick R, Moses H. Transforming growth factor-l3. A family of growth regulatory peptides. Ann NY Acad Sci 1990;593:208-17. 3. Centrella M, Massague J, Canalis E. Human platelet-derived transforming growth factor-(3 stimulates parameters of bone growth in fetal rat calvaries. Endocrinology 1986; 119:2306-12. 4. Thompson NL, Flanders KC, SmithJM, Ellingsworth LR, Roberts AB , Sporn MB . Expression of transforming growth factor-l3, in specific cells and tissues of adult and neonatal mice. J Cell Bioi 1989;108:661-8.
Transforming growth factor-l3t expression in human placenta
857
5. Krummel TM, Michna BA, Thomas BL, et al. Transforming growth factor 13 (TGF-I3) induces fibrosis in a fetal wound model. J Pediatr 1988;22:647-82. 6. Mustoe T, Pierce G, Thomason A, Gramates P, Spron M, Deuel T. Accelerated healing of incisional wounds in rats induced by transforming growth factor-l3. Science 1987;237: 1333-6. 7. Masui T, Wakefield L, Lechner J , Laveck M, Harris C, Sporn M. Type-13 transforming growth factor is the primary differentiation-inducing serum factor for normal human bronchial epithelial cells. Proc Nat! Acad Sci USA 1986;83:2438-42. 8. De Benedetti F, Falk LA, Ellingsworth LR, Ruscetti FW, Faltynek CR. Synergy between transforming growth factor-13 and tumor necrosis factor-a in the induction of monocytic differentiation of human leukemic cell lines. Blood 1990;75:626-32. 9. Berchuck A, Olt G, Everitt L, Soisson A, Bast R, Boyer C. The role of peptide growth factors in epithelial ovarian cancer. Obstet Gynecol 1990;75:255-62. 10. Pijnenborg R, Bland JM, Robertson WB, Dixon G, Brosens I. The pattern of interstitial trophoblastic invasion of the myometrium in early human pregnancy. Placenta 1981;2:303-16. 11. Chomzynski P, Sacchi E. Single step method of RNA isolation by quanidium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987;162:156-9. 12. Midgley A, Pierce G, Deneau G, Gosling]. Morphogenesis of syncytiotrophoblast in vivo: an auto radiographic demonstration. Science 1963; 141 :349-50. 13. Winick M, Cosicu A, Noble A. Cellular growth in human placenta. I. Normal placental growth. Pediatrics 1967;39:248-51. 14. HuntJS, Soares MJ, Lei MG, et al. Products oflipopolysaccharide-activated macrophages (tumor necrosis factora , transforming growth factor-l3) but not lipopolysaccharide modify DNA synthesis by rat trophoblastic cells exhibiting the 80-kDA lipopolysaccharide-binding protein. J Immunol 1989;143:1606-13.
Key words: Transforming growth factor-I3., human placenta
Placental implantation and subsequent growth and development have been described asa pseudomalignant process. Similarities between trophoblastic cell growth and malignant cells include invasiveness, rapid cell proliferation, lack of cell contact inhibition, and a degree of immune privilege. These malignant characteristics of placental development appear to be strictly regulated throughout normal gestation, because placental growth is limited in both its extent and duration. In fact, during embryogenesis and placentation tissues and organs develop in a sequential pattern, implying that genes coding for particular proteins are turned on and off in a precise and well-regulated manner. It is now becoming apparent that growth inhibitors play an important role in maintaining tissue homeostasis. Peptide growth factors, active in common regulatory pathways, may be important regulators of placental develo~ment.
-Transforming growth factor-l3. (TGF-I3.) is a polyFrom the Division of Maternal-Fetal Medicine, Departments of Obstetrics and Gynecology,' and the Department of Anatomy and Cell Biology,' University of Cincinnati College of Medicine. Supported in part by a Research Grant to the Department of Obstetrics and Gynecology, University of Cincinnati College of Medicine, Cincinnati, Ohio. Presented at the Eleventh Annual Meeting of the Society of Perinatal Obstetricians, San Francisco, California, January 28-February 2,
1991. Reprint requests: Lauren]. Dungy, MD, Division of Maternal-Fetal Medicine, Department of Obstetrics and Gynecology, University of Cincinnati College of Medicine, 231 Bethesda Ave., Cincinnati, OH 45267-0526.
6/6/30897
peptide originally identified in tumor cells and postulated to be involved in transformation and neoplastic growth. Its presence in numerous tissues,I-4 however, implies a normal physiologic role that appears to be multifunctional. TGF-131 has been shown to stimulate growth of some cell types but to inhibit growth of others, to stimulate growth of fibroblasts and promote wound healing," 6 and to induce differentiation of leukemic and bronchial epithelial cell lines. 7• S A fundamental property of TGF-131 is its ability to inhibit proliferation of both normal and malignant cell lines. TGF13. has been shown to inhibit growth of different ovarian cancer cell lines, as well as some human leukemic cell lines. s . g As such, we hypothesized that TGF-I3. may play an important role in the inhibition of trophoblastic invasion of the myometrium during placental development. In humans, this process is largely limited to the eighth week through the eighteenth week of gestation. 10 We evaluated the temporal expression of TGF-131 in human placenta to begin to examine this hypothesis.
Material and methods Collection of placental tissue. Placental tissue was obtained after curettage, spontaneous delivery, or cesarean section and immediately placed in liquid nitrogen. Informed consent was obtained from patients in all instances, in accordance with the protocol approved by our institutional review board. Time frames of placental tissue studied were chosen to correlate with early, mid, and late gestation. Placental tissue obtained from a total of 20 patients was used for analysis. Initially, a
853
854
Dungy, Siddiqi, and Khan
Onobcr 199 1 Am .l Ollslel Gmccol
400,---~--------------------,
w
300
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Z
m a:
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34
WEEKS GESTATION Fig. 1. Graph showing results of scanning densitometry of blots of five representative RNA samples. TGF-~ , expression is noted throughout gestational period shown , with peak expression at 17 and 34 weeks' gestation.
panel of total ribonucleic acid (RNA) from placental tissues ranging from 6 to 40 weeks' gestation was studied with Northern blot analysis. These studies were repeated with total RNA from different placental tissues of similar gestational ages . Three panels of RNA were studied. Additionally, r\orthern blot analysis was performed with 100 f.Lg of ' <" .J RNA from placental tissue of IS through 19 weeks' gestational age and 32 through 34 weeks' gestational age to furt her evaluate time frames where peak ex :) 1 ession had previously been identified. RNA isolation. Total RNA was isolated from the placental tissues according to the technique of Chomzynski and Sacchi." Briefly, approximately 2 gm of placental tissue was homogenized in 10 ml extractio n solution (4 mol/L guanidine thiocyanate ; 25 mmol / L sodium citrate, pH 7.0; 0 5. % sarcosyl; 0.1 mol/L 2-j)-mercaptoethanol ; 2 mol/L sodium acetate; and 10 ml phenol). The homogenate was mixed with chloroform / isoamyl alcohol (2 ml of 49: 1 solution) and chilled on ice for 15 minutes. After centrifugation for 20 minutes at 10,000 g at 4° C, the aqueous phase was removed and precipitated with one volume of isopropanol at - 20° C for 2 hours. This was fo llowed by a second centrifugation for 20 minutes at 10,000 g at 4° C. The resultant pellet was dissolved in extraction solution (600 f.LI) and precipitated with isopropanol (I : I,vol/ vol) and sodium acetate (I: 10, vol/vol) at -20° C for approximately 2 hours. After centrifugation for 10 minutes at 4° C, the pellet was washed with 70 % ethanol and vacuum dried for approximately 5 minutes. The pellet was then resuspended in diethylprocarbonate-treated water. Total RNA concentration was quantitated by absorption spectrophotometry at 260 nm. Northern blot analysis. RNA samples (100 f.Lg) were
resolved by electrophoresis on formaldehyde agarose gels and transferred to nitrocellulose membranes by towel blotting. The integrity and quantity of each RNA sample were ensured by stainin g the ge l with ethidium bromide and visualizing the gel under ultraviolet transillumination. The filters were prehybridized in hybridization solution containing 10 X Denhardt's solution , 0.1 % sod ium dodedecyl sulfate, 100 f.Lg / ml single standard sperm deoxyribonucleic acid, 50% deionized forma mide, and a 5 x solution of 3 mollL sodium chloride, 0.1 mol/L sodium phosphate, 0.02 mol/L ethylenediaminetetraacetic acid, and sodium hydroxide and then hybridized in the same solution containing a phosphorus 32-labeled human TGF-j), complementary deoxyribonucleic acid probe ( I x 10'; counts / min / ml) at 42° C overnight. After four washings (final wash I X saline sodium citrate buffer with 0.1 % sodium dodedecyl sulfate at 55° C for 20 minutes) the filters were subjected to autoradiography. The position of 18S and 28S RNA was determined, and the single band observed, which corresponded to the position of TGF-f3, messenger RNA (mRNA) (2.1 kb), was quantitated by scanning densitometry with a Hoefer GS 300 (Hoefer Scientific Instruments, San Francisco) gel scanner. Our initial studies with 25 and 50 f.Lg of total RNA revealed expression only at 17 and 34 wee ks' gestation . In later blots TGF-f3, expression was noted at all time points when 100 f.Lg of total RNA was used. For this reason 100 f.Lg samples were used in all experiments reported in this study. Immunohistochemical analysis. Placental sections (10 f.Lm) from 17 and 34 weeks' gestation were fixed in chi lled methanol, followed by fixation in sodium periodate, lysine, and paraformaldehyde solution. The sections were then incubated at room temperature with a phosphate-buffered saline solution containing bovine serum albumin (1%) and 0.1 % Triton-X-lOO (Fischn Scientific, Fair Lawn , N.J .) compound for 1O minutes, followed by a 30-minute incubation with phosphatebuffered saline solution containing 0.2% gelatin to minimize background contamination. The sections were then incubated with polyclonal rabbit antibody against human TGF-f3! (1: 50 dilution in phosphate-buffered saline solution-bovine serum albumin) for I hour at 37° C. TGF-f3, antibody used in this study was obtained from Collage n Corporation, Palo Alto, Calif. After three washings with phosphate-buffered saline solution containing 0.2% gelatin fO!' 15 minutes each, the specimens were incubated with fluorescein-conjugated goat anti-rabbit polyclonal antibody (I : 50 dilution in phosphate-buffered saline solution -bovine serum albumin) for 45 minutes at 37° C. After three washings in phosphate-buffered saline solution containing 0.2% gelatin, immunofluore·scent sites were visualized in an epiflu-
Voillme 16!\ NlImber 4. Pal'! I
Transforming growth factor-I3, expression in human placenta
855
Fig. 2. Northern blot of total RNA samples isolated from placental tissues near midgestation. TGF13, expression is noted throughout (15 to 19 weeks). Maximal expression was found at 17 weeks' gestation by densitometric quantitation.
orescence microscope (Leitz, Wetzler, Germany) . Sequential sections were treated in a similar manner with nonimmune rabbit serum substituted at the same concentration for TGF-f3, antibody to serve as control. Results
With Northern blot analysis and 100 fJ..g of total RNA, we were able to identify TGF-f3, mRNA expression throughout the gestational ages studied (6 through 40 weeks). Fig. 1 represents the continuous scanning densitometry graph of a blot of equal amounts of total RNA isolated from placental tissue ranging from 9 to :H weeks' gestation. Expression of TGF-f3, was noted throughout the time points examined, with peaks of expl'ession noted at 17 and 34 weeks' gestation. This is consistent with our initial findings of TGF-f3, mRNA expression only at 17 and 34 weeks' gestation using lower amounts of total RNA (25 and 50 fJ..g). Northern blot analysis of total RNA isolated from tissue samples ranging from 15 to 19 weeks' gestation revealed TGF-f3, expression throughout these time points (Fig. 2). Maximal expression was observed at 17 weeks by densitometric quantitation. Analysis of total RNA isolated from tissue of 32 to 34 weeks' gestation revealed minimal expression in 32 and 33 week samples with a striking increase of expression occurring at 34 weeks (Fig. 3). We have looked at the expression of c-jun and jun-B in human placenta. These protooncogenes have been associated with cellular growth processes, including both proliferation and differentiation. Their patterns of expression did not correspond to that found with TGF-f3, (unpublished data). This would imply that the
Fig. 3. Northern blot of LOtal RNA samples of placental tissue in late gestation. Minimal expression occurred at 32 and 33 weeks with increased expression at 34 weeks.
increased expression of TGF-f3, near 17 and 34 weeks' gestation is not related to a general phenomenon. After identification of peak TGF-f3, mRNA expression in 17 and 34 weeks' gestational age placental tissue, we used immunohistochemical analysis to localize TGFf3, protein expressinn in these tissues. TGF-f3, protein expression was localized to the periphery of placental villi corresponding with the syncytiotrophoblastic layer (Fig. 4). There was an absence of staining of cell types of the villous core. Control specimens did not show this pattern of staining.
856
Dungy, Siddiqi, and Khan
October 1991 Am J Obstet Gynecol
Fig. 4. Immunohistochemical analysis of 17-week placental tissue. TGF-I3, protein expression is localized to syncytiotrophoblastic layer only. (Original magnification x 100.)
Comment The placenta is a very dynamic organ, passing through stages of graft acceptance, cell differentiation, invasive growth, and cessation of both growth and invasiveness. Knowledge about regulatory factors involved in these processes is limited. TGF-J3, promotes proliferation of some cell types but inhibits proliferation of others, depending on cell conditions and concentration. A fundamental property is its ability to inhibit proliferation of both normal and malignant cell lines. The observations made in our study suggest that TGF-J3, is expressed in human placenta throughout gestation. Additionally, two peaks of expression were noted: an initial peak occurring near midgestation at 17 weeks and a second peak in later gestation, near 34 weeks. In humans trophoblastic invasion of the myometrium is largely limited to the eighth through eighteenth weeks of gestation. Large numbers of cytotrophoblastic cells invade the decidua basalis and underlying myometrium. Fusion of cytotrophoblastic cells results in formation of syncytial cells. An absence of mitotic activity is noted in the syncytiotrophoblast.'2 Increased mRNA expression of TGF-J3, near 17 weeks' gestation suggests that it may playa role in inhibition of myometrial trophoblastic invasion. Also, TGF-J3, protein expression was localized to the syncytiotrophoblastic layer with immunohistochemical analysis.
The expression ofTGF-J3" a factor known to be related to inhibition of cell proliferation, in these mitotic endstage cells supports its possible role in the inhibition of cytotrophoblastic myometrial invasion. It is possible that different trophoblastic cell types may express TGFJ3, near midgestation and be involved in myometrial invasion. Our studies, however, were able to identify only TGF-J3, protein expression localized to the villous syncytiotrophoblast with an absence of staining of other cell types. Although we cannot rule out decidual contamination as a source ofTGF-J3, mRNA expression in our specimens, immunohistochemical analysis identified TGF-J3, protein expression only in the syncytiotrophoblastic layer. Absolute growth of human placenta as determined by deoxyribonucleic acid content declines in late gestation beginning at 35 to 36 weeks' gestation, at which time cytotrophoblastic proliferation essentially ceases. 13 TGF-J3, has been shown to inhibit rat trophoblastic cell deoxyribonucleic acid synthesis in a dose-dependent manner.'1 A second peak of TGF-J3, expression near 34 weeks' gestation may therefore be related to this cessation of cytotrophoblastic cell proliferation and decline in placental growth. This was again supported by localization of TGF-I3, protein expression in the syncytiotrophoblastic layer with immunohistochemical analysis. In summary, with Northern blot analysis TGF-I3,
Volume 165 Number 4, Part 1
expression was noted throughout gestation in our study. Peak expression was noted at 17 and 34 weeks' gestation. Immunohistochemical staining revealed TGF-I3, expression restricted to the syncytiotrophoblastic layer. TGF-I3, expression in the syncytiotrophoblast at these time points may be related to inhibition of cytotrophoblastic proliferation occurring at these stages of placental development. Pathologic conditions of trophoblastic in vas ion exhibited in gestational trophoblastic disease and choriocarcinoma represent a deviation from normal trophoblastic growth. We have not examined such tissues. Examination of these tissues with comparison to findings in nonpathologic conditions would be of value in further elucidation of the role of TGF-I3, in regulating trophoblastic myometrial invasion. REFERENCES 1. Sporn M, Roberts A. The transforming growth factorbetas: past, present and future. Ann NY Acad Sci 1990;593: 1-6. 2. Miller D, Pelton R, Deryick R, Moses H. Transforming growth factor-l3. A family of growth regulatory peptides. Ann NY Acad Sci 1990;593:208-17. 3. Centrella M, Massague J, Canalis E. Human platelet-derived transforming growth factor-(3 stimulates parameters of bone growth in fetal rat calvaries. Endocrinology 1986; 119:2306-12. 4. Thompson NL, Flanders KC, SmithJM, Ellingsworth LR, Roberts AB , Sporn MB . Expression of transforming growth factor-l3, in specific cells and tissues of adult and neonatal mice. J Cell Bioi 1989;108:661-8.
Transforming growth factor-l3t expression in human placenta
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