Estrogen mediates mammary epithelial cell proliferation in serum-free culture indirectly via mammary stroma-derived hepatocyte growth factor

Epithelial-stromal cell interactions are important for normal development and function of the mouse mammary gland. The steroid hormone estrogen is required for epithelial cell proliferation and ductal development in vivo. Recent studies of estrogen receptor alpha knockout mice indicate that estrogen-induced proliferation is dependent upon the presence of estrogen receptor in mammary stromal cells, but not in epithelial cells. The purpose of the present study was to identify the underlying mechanism of estrogen-dependent stroma-derived effects on mammary epithelium. We have developed a minimally supplemented serum-free medium, collagen gel primary mammary coculture system to address the issue of stroma-derived, estrogen-dependent effects on epithelial cell proliferation. Conditioned medium from mammary fibroblasts or coculture with mammary fibroblasts caused increased epithelial cell proliferation and produced tubular/ductal morphology. Hepatocyte growth factor (HGF) was identified as the mediator of this effect, as the proliferative activity in fibroblast-conditioned medium was completely abolished by neutralizing antibody to HGF, whereas neutralizing antibodies to either epidermal growth factor or IGF-I had no effect. Treatment of mammary fibroblasts with estrogen increased the production of HGF. From these results we conclude that estrogen may indirectly mediate mammary epithelial cell proliferation via the regulation of HGF in mammary stromal cells and that HGF plays a crucial role in estrogen-induced proliferation in vivo.     

Epidermal growth factor stimulates cell proliferation and inhibits functional differentiation of mouse mammary epithelial cells in culture

Mouse mammary epithelial cells cultured on collagen gels multiplied and produced casein and alpha-lactalbumin in response to insulin, cortisol, and PRL. The addition of epidermal growth factor (EGF) at 50 ng/ml increased the total number of epithelial cells by 30-40% and thymidine incorporation into DNA 4.7-fold after 5 days of culture. In contrast, EGF inhibited hormonal induction of the synthesis of casein and alpha-lactalbumin in those cells by about 45% and 55%, respectively, without inhibiting total protein synthesis. Furthermore, EGF decreased casein mRNA activity by 55% and increased total mRNA activity by 66% in cells cultured with the three hormones. These effects of EGF were apparent at 0.1 ng/ml and were maximal at 50-100 ng/ml and could be reversed by its removal from the medium, followed by the addition of anti-EGF antibody. The inhibition of casein synthesis by EGF was unaffected by the concentrations of insulin, cortisol, and PRL. Other growth factors, such as fibroblast growth factor, multiplication-stimulating activity, nerve growth factor, and platelet-derived growth factor, did not simulate the effects of EGF. Cytarabine (1 microgram/ml), which inhibited thymidine incorporation into DNA by 94%, did not block the inhibitory action of EGF on casein synthesis. These results suggest that EGF serves as a regulator of hormone-dependent growth and differentiation of mammary epithelial cells.     

Hepatocyte growth factor is required for progestin-induced epithelial cell proliferation and alveolar-like morphogenesis in serum-free culture of normal mammary epithelial cells

The steroid hormones, estrogen and progesterone, are required for mammary epithelial cell proliferation and alveolar morphogenesis in vivo. We have developed a minimally supplemented, serum-free medium, collagen gel primary mammary culture system to determine the mechanism of progestin-induced proliferation and alveolar morphogenesis. In epithelial cells cultured alone, treatment with progestin (R5020) alone produced a lumen within the epithelial organoids, but did not stimulate epithelial cell proliferation. The formation of lumens was associated with increased apoptosis, targeted within the organoids. We have previously reported that in our culture system hepatocyte growth factor (HGF) increases epithelial cell proliferation and induces a tubulo-ductal morphological response. In the present report we show that treatment with HGF and progestin (R5020) further increases epithelial proliferation above that with HGF alone and also produces an alveolar-like morphology similar to that observed in vivo in response to progestin treatment. To the best of our knowledge this is the first in vitro demonstration of both progestin-induced proliferation and alveolar-like morphogenesis of normal nonpregnant mouse mammary epithelial cells in vitro. These results suggest that HGF may play a crucial role in progestin-induced proliferation and morphogenesis in vivo.     

Stimulation of DNA synthesis in cultures of ovine mammary epithelial cells by insulin and insulin-like growth factors

Ovine mammary epithelial cell clumps (30-90 microns) were plated onto attached gels of rat tail collagen in serum-free medium. Synthesis of DNA by these cultures could be stimulated by insulin-like growth factor-I (IGF-I) with a median effective dose of 5 micrograms/l, irrespective of stage of pregnancy. The time-course of response, however, was significantly slower in cells prepared from mammary tissue of non-pregnant and early pregnant sheep compared with sheep later in pregnancy. IGF-II had approximately 10% of the potency of IGF-I in stimulating DNA synthesis. Insulin acted over a wide concentration range and produced a maximum rate of stimulation not significantly different from that produced by IGF-I. These results are consistent with actions through the type-I IGF receptor although insulin may also act through its own receptor, possibly stimulating local IGF-I production. It is concluded that IGF-I is an important mitogen for ovine mammary epithelial cells.     

Stimulation of mammary epithelial cell growth in vitro: interaction of epidermal growth factor and mammogenic hormones

A serum-free primary cell culture system was used to examine the direct effects and interactions of mammogenic hormones and epidermal growth factor (EGF) on the growth of mouse mammary epithelial cells. Epithelial cells were isolated by collagenase dissociation followed by Percoll gradient centrifugation and cultured within collagen gels in a mixture of Ham's F-12-Dulbecco's Minimum Essential Medium (1:1) containing insulin (10 micrograms/ml), crude soybean lecithin, trace elements, trypsin inhibitor, and antioxidants. Progesterone (P; 10(-6) - 10(-8) M) or ovine PRL (1 microgram/ml), in the absence of EGF, stimulated the growth of cells from mature virgin mice 2- to 4-fold over that of controls cultured in basal medium only. P and PRL synergized in stimulating growth 3- to 17-fold. 17 beta-Estradiol (10(-7) - 10(-10) M) alone did not stimulate growth or synergize with P and/or PRL. This lack of growth stimulation by 17 beta-estradiol was also observed in medium containing a low concentration of insulin (0.1 microgram/ml). EGF (10 ng/ml) alone stimulated growth to the same extent as the combination of P and PRL. EGF at 1, but not 10, ng/ml when combined with P and PRL could additively stimulate growth. Cells from midpregnant mice were less responsive than cells from virgin mice to the growth-stimulating effects of the combination of P and PRL (2-fold stimulation at most), but not to EGF (3- to 6-fold stimulation). Corticosterone, deoxycorticosterone, and aldosterone, but not cortisol, could synergize with PRL in stimulating the growth of cells from mature virgin mice. However, only deoxycorticosterone could stimulate growth in the absence of PRL. These results suggest that PRL, P, and adrenal corticoids may directly stimulate the growth of mouse mammary epithelial cells. The physiologically relevent adrenal corticoids, corticosterone and aldosterone, only potentiate the stimulatory effect of PRL. The hormonal stimulation of growth in vitro can be obscured by an optimum concentration (10 ng/ml) of EGF. The relative growth responses to mammogenic hormones and EGF may depend on the degree of differentiation of the cells.     

酒精通过抑制肠上皮干细胞增殖损伤肠上皮的更新修复能力

目的 研究酒精对肠上皮干细胞(ISC)和肠上皮更新修复能力的影响。方法 将18只C57BL/6小鼠随机分为对照组(n=9)和酒精处理组(n=9)。采用Gao-Binge法制备慢性酒精中毒模型。在造模成功后,腹腔注射5-溴-2-脱氧脲苷(BrdU),分别在注射后2 h、24 h和72 h取小肠组织,采用免疫组化法检测BrdU阳性细胞和ISC特异性标志物Lgr5表达。结果 与对照组小鼠比,酒精处理组小鼠小肠绒毛高度显著缩短、萎缩;酒精处理组小鼠ISC细胞Lgr5表达显著弱于对照组;酒精处理组小鼠每个肠隐窝BrdU阳性细胞数量为(3.50±0.65)个/肠隐窝,显著少于对照组【(7.90±1.08)个/肠隐窝,P＜0.05】;在注射BrdU 后2 h、24 h和72 h,酒精处理组小鼠小肠BrdU阳性细胞迁移距离分别为(66.67±1.60)μm、(219.40±12.11)μm和(313.90±9.76)μm,显著短于对照组【分别为(111.10±1.60)μm、(319.00±10.04)μm和(625.90±3.34)μm,P＜0.05】。结论 酒精通过抑制ISC引起肠上皮细胞增殖和迁移能力下降,从而损伤肠上皮的更新修复能力,导致肠上皮屏障功能障碍。     

Influencing factors of rat small intestinal epithelial cell cultivation and effects of radiation on cell proliferation

Crypt epithelial cells in normal small intestineproliferate at a high speed. But they are verydifficult to culture in vitro and passage stably. A lotof studies have been done. Some domestic labsisolated and cultured crypt cells from embryonalintestines and aseptic animal intestine, but failed.We introduced normal rat epithelial cell line-IEC-6from the USA and its living condition for stablepassage was successfully established after trials. Thecell line was testified to be the small intestinal     

Influencing factors of rat small intestinal epithelial cell cultivation and effects of radiation on cell proliferation

INTRODUCTIONCrypt epithelial cells in normal small intestineproliferate at a high speed. But they are verydifficult to culture in vitro and passage stably. A lotof studies have been done[1-16]. Some domestic labsisolated and cultured crypt cells from embryonalintestines and aseptic animal intestine, but failed.We introduced normal rat epithelial cell line-IEC-6from the USA and its living condition for stablepassage was successfully established after trials. Thecell line was testified to be the small intestinalepithelial cell by electron microscopy,immunihistochemistry and enzymatic histoch-emistry. It has been applied to some relatedresearch work[17-21]. It was found that manyfactors were involved in the culture system. Ourpresent study focuses on the culture method and theinfluencing factors on IEC-6.     

Regulation of rat mammary epithelial cell proliferation and differentiation by tumor necrosis factor-alpha.

The effect of tumor necrosis factor-alpha (TNF alpha) on the growth and differentiation of normal rat mammary epithelial cells was evaluated using a model system in which cells were grown within a reconstituted basement membrane under defined serum-free medium conditions. TNF alpha (5-10,000 U/ml) stimulated mammary epithelial cell proliferation both under conditions previously considered optimal for their growth as well as in medium deficient in epidermal growth factor (EGF). Moreover, TNF alpha could completely substitute for EGF for cell proliferation. Under optimal conditions, TNF alpha had no effect on morphological differentiation, but in medium either lacking or deficient in EGF or when suboptimal reconstituted basement membrane was used, TNF alpha (5-100 U/ml) had a marked stimulatory effect on lobular and ductal morphogenesis. The effect of TNF alpha on functional differentiation, as assessed by casein production, was more complex. In optimal lactogenic medium, TNF alpha (10-10,000 U/ml) inhibited casein production. In the absence of EGF, however, the effect of TNF alpha appeared to follow a bell-shaped curve. Thus, omission of EGF per se resulted in a marked suppression of casein production, possibly secondary to an inhibition of morphological development. At low concentrations (approximately 5 U/ml), TNF alpha stimulated casein production in parallel to its stimulation of morphological differentiation, although not to the same extent as in medium containing optimal levels of EGF. However, once maximal stimulation of morphogenesis had been achieved, further increasing the TNF alpha concentration from 5 to 100 U/ml resulted in a concentration-dependent inhibition of casein production. This suggests that TNF alpha may have a direct inhibitory effect on casein gene expression. In summary, this is the first study to report that the multifunctional cytokine TNF alpha is a potential regulator of the growth and development of the mammary gland.     

Suppressor of cytokine signaling-2: a growth hormone-inducible inhibitor of intestinal epithelial cell proliferation

BACKGROUND & AIMS: Growth hormone (GH) and insulin-like growth factor-I (IGF-I) increase intestinal growth. GH is thought to act indirectly via IGF-I. In several models, including rats given total parenteral nutrition (TPN), IGF-I more potently stimulates mucosal growth than GH, even when GH induces similar circulating IGF-I levels. These studies test the hypothesis that GH induces a suppressor of cytokine signaling (SOCS), which inhibits intestinal epithelial cell (IEC) proliferation. METHODS: Rats on TPN received vehicle, GH, or IGF-I. Jejunal SOCS (SOCS-1, -2, -3, and cytokine-inducible SH2-domain-containing protein [CIS]) and IGF-I messenger RNA (mRNA) were quantified. Caco-2, IEC-6 cells, and SOCS-2 null and wild-type (WT) mice were used to examine the expression and functional role of SOCS-2. RESULTS: As reported previously, IGF-I, but not GH, prevented mucosal atrophy during TPN, although GH elevated plasma IGF-I and increased body weight. GH, but not IGF-I, induced jejunal SOCS-2 mRNA. SOCS-2 mRNA levels in GH and IGF-I-treated rats inversely correlated with mucosal weight. SOCS-2 is expressed in Caco-2 cells, and elevated SOCS-2 expression in postconfluent cells is associated with reduced proliferative rates. SOCS-2 overexpression in Caco-2 cells inhibited cell proliferation and promoted differentiation. In IEC-6 cells, GH induced SOCS-2 and reduced basal or IGF-I-induced proliferation. GH also reduced proliferative activity in isolated crypts from WT but not SOCS-2 null mice, and SOCS-2 null crypts showed enhanced proliferative responses to GH and IGF-I. SOCS-2 null mice have increased intestinal weight and length. CONCLUSIONS: SOCS-2 is a GH-inducible, novel inhibitor of intestinal epithelial cell proliferation and intestinal growth.     

Novel insights into human intestinal epithelial cell proliferation in health and disease using confocal microscopy.

Measurement of intestinal epithelial cell proliferation has provided important information concerning tissue responses in neoplasia, enteropathy, and adaptation. This study reexamined current concepts regarding intestinal proliferation by using a novel confocal microscopical technique to map mitotic figures accurately within the intact three dimensional framework of the crypts of Lieberkühn. The ability of confocal microscopy to simultaneously measure crypt morphology and internal detail, without disrupting spatial cell arrangements, has provided important new data. These question the ability of existing methods to accurately measure and interpret proliferative changes in the gut. This work investigates intestinal proliferation in children with coeliac disease, a well defined hyperproliferative disorder, in comparison with the healthy intestine. These results show that crypt cell division occurs with an equal probability in health and disease. In addition, increased crypt cell production rates are largely caused by a change in crypt size rather than a change in cell cycle time or crypt growth fraction and, as such, alter our understanding of kinetic responses in gastrointestinal disease.     

Regulation of intestinal epithelial cell growth by transforming growth factor type beta.

A nontransformed rat jejunal crypt cell line (IEC-6) expresses transforming growth factor type beta 1 (TGF-beta 1) mRNA, secretes latent 125I-labeled TGF-beta 1 competing activity into culture medium, and binds 125I-labeled TGF-beta 1 to specific, high-affinity (Kd = 3.7 pM) cell surface receptors. IEC-6 cell growth is markedly inhibited by TGF-beta 1 and TGF-beta 2 with half-maximal inhibition occurring between 0.1 and 1.0 ng of TGF-beta 1 per ml. TGF-beta 1-mediated growth inhibition is not associated with the appearance of biochemical markers of enterocyte differentiation such as alkaline phosphatase expression and sucrase activity. TGF-beta 1 (10 ng/ml) increases steady-state levels of its own mRNA expression within 8 hr of treatment of rapidly growing IEC-6 cells. In freshly isolated rat jejunal enterocytes that are sequentially eluted from the crypt villus axis, TGF-beta 1 mRNA expression is most abundant in terminally differentiated villus tip cells and least abundant in the less differentiated, mitotically active crypt cells. We conclude that TGF-beta 1 is an autoregulated growth inhibitor in IEC-6 cells that potentially functions in an autocrine manner. In the rat jejunal epithelium, TGF-beta 1 expression is most prominently localized to the villus tip--i.e., the region of the crypt villus unit that is characterized by the terminally differentiated phenotype. These data suggest that TGF-beta 1 may function in coordination of the rapid cell turnover typical for the intestinal epithelium.     

Growth factor- and cyclic nucleotide-induced proliferation of normal and malignant mammary epithelial cells in primary culture

Sustained growth of normal mouse mammary epithelial cells in primary culture, leading to an increase in cell number, in response to growth factors [epidermal growth factor (EGF) and fibroblast growth factor (FGF)] or cholera toxin has been achieved by embedding the cells inside collagen cells. Inclusion of agents known to increase the level of cellular cAMP have been found to be favorable for mammary epithelial cell proliferation. Cholera toxin is by far the best of all of the agents tested (prostaglandins E1 and E2, isoproterenol, theophylline, and dibutyryl cAMP). When growth factors (EGF or FGF) are added with cholera toxin, a synergistic effect resulting in a response much greater than with either of them alone is seen. This synergism was best seen in normal mammary epithelial cells from nonpregnant mice. The extent of this synergistic effect was found to be less in normal cells from pregnant mice, suggesting that these cells may be less responsive to EGF during pregnancy. Tumor cells were found to be rather inconsistent in their responses to EGF and cholera toxin, ranging from a minimal response, similar to that of normal cells from pregnant animals, to a maximal response, similar to that of normal cells from nonpregnant animals.     

Tumor promoter 12-O-tetradecanoylphorbol 13-acetate, like epidermal growth factor, stimulates cell proliferation and inhibits differentiation of mouse mammary epithelial cells in culture.

12-O-Tetradecanoylphorbol 13-acetate (TPA) is a potent tumor promoter and shares several biological activities of epidermal growth factor (EGF). Recently we have shown that EGF stimulates DNA synthesis and inhibits milk protein synthesis induced by insulin, cortisol, and prolactin in a primary mouse mammary epithelial cell culture [Taketani, Y. & Oka, T. (1983) FEBS Lett., in press]. Using this system, we examined the biological action of TPA in reference to that of EGF. TPA stimulated cell proliferation and inhibited the synthesis of milk proteins casein and alpha-lactalbumin during a 5-day culture. The ED50 was about 2.5 ng/ml for DNA synthesis and 0.1 ng/ml for milk protein synthesis. Removal of TPA from the medium normalized DNA synthesis completely and casein synthesis partially. The ability of various TPA analogs to stimulate DNA synthesis and inhibit casein synthesis correlated with their potency as tumor promoters. Both TPA and EGF decreased the specific prolactin binding of cultured cells by approximately equal to 50%. These results indicate that TPA, like EGF, switches the developmental course of mammary epithelium from differentiation to proliferation.     

Prolactin regulates mammary epithelial cell proliferation via autocrine/paracrine mechanism

Prolactin (PRL) is essential for a number of developmental events in the mammary gland. Work with PRL and PRL receptor knockout mice has shown that PRL indirectly regulates ductal side branching during puberty and directly controls lobuloalveolar development and lactogenesis during pregnancy. Anterior pituitary or placental PRL is thought to be responsible for these functions via an endocrine mechanism; however, PRL is also produced in a number of extrapituitary sites including the mammary gland. The physiologic relevance of mammary PRL remains unknown. In this study we utilized mammary recombination in Rag1^−/− hosts, to determine whether mammary PRL plays a role in the regulation of mammary gland development. Mammary glands formed with the PRL gene deleted from either the epithelium, stroma, or both displayed normal development, on the basis of whole mount and hematoxylin and eosin histology, during puberty and lactation. At the end of pregnancy, a 2.8-fold decrease in bromodeoxyuridine incorporation was observed in the epithelial cells of mammary glands formed using PRL knockout epithelium compared with those formed using wildtype epithelium. No balancing alteration in the rates of apoptosis was detected. Thus, mammary-derived PRL influences mammary epithelial cell proliferation via an autocrine/paracrine mechanism, establishing a physiologic function for mammary PRL during mammopoiesis.