Reversible activation of mouse metal response element-binding transcription factor 1 DNA binding involves zinc interaction with the zinc finger domain.

The DNA-binding activity of the Zn finger protein metal response element-binding transcription factor 1 (MTF-1) was rapidly induced both in vivo in mouse Hepa cells, canine MDCK, and human HeLa cells after incubation in medium containing zinc and in vitro in whole-cell extracts to which zinc was added. Acquisition of DNA-binding capacity in the presence of free zinc was temperature and time dependent and did not occur at 4 degrees C. In contrast, activated MTF-1 binding to the metal response element occurred at 4 degrees C. After Zn activation, mouse MTF-1 binding activity was more sensitive to EDTA and was stabilized by DNA binding relative to the Zn finger transcription factor Sp1. After dilution of nuclear or whole-cell extracts from Zn-treated cells and incubation at 37 degrees C, mouse MTF-1 DNA-binding activity was no longer detected but could be completely reconstituted by the subsequent readdition of zinc. In vitro-synthesized, recombinant mouse MTF-1 displayed a similar, reversible temperature- and Zn-dependent activation of DNA-binding activity. Analysis of deletion mutants of recombinant MTF-1 suggests that the Zn finger domain is important for the Zn-dependent activation of DNA-binding capacity. Thus, mouse MTF-1 functions as a reversibly activated sensor of free zinc pools in the cell.

The heat shock response in HeLa cells is accompanied by elevated expression of the c-fos proto-oncogene.

Several known inducers of the heat shock response (heat stress, arsenite, and heavy metals) were shown to cause a significant elevation of c-fos mRNA in HeLa cells. Heat stress resulted in a time- and temperature-dependent prolonged elevation in the level of c-fos mRNA, which was accompanied by increased translation of c-fos protein and its appearance in the nucleus. Elevated expression of c-fos during heat stress was paralleled by induction of hsp 70 mRNA, while levels of c-myc and metallothionein mRNAs declined. Treatment of HeLa cells with arsenite or heavy metals also resulted in increased levels of hsp 70, as well as c-fos mRNA. Although elevated expression of c-fos was prevented by inhibitors of RNA synthesis, analysis of relative rates of gene transcription showed that during heat stress there was a negligible change in c-fos transcription. Therefore, the enhanced expression of c-fos during the heat shock response is likely to occur primarily through posttranscriptional processes. Cycloheximide was also shown to significantly increase the c-fos mRNA level in HeLa cells. There results are consistent with the observation that these inducers of the heat shock response, as well as cycloheximide, repress protein synthesis and suggest that the increase in the level of c-fos mRNA is caused by an inhibition of protein synthesis. This supports the hypothesis that c-fos mRNA is preferentially stabilized under conditions which induce the heat shock response, perhaps by decreased synthesis of a short-lived protein which regulates c-fos mRNA turnover.

Activation of metallothionein gene expression by hypoxia involves metal response elements and metal transcription factor-1.

Metallothioneins (MTs) are a family of stress-induced proteins with diverse physiological functions, including protection against metal toxicity and oxidants. They may also contribute to the regulation of cellular proliferation, apoptosis, and malignant progression. We reported previously that the human (h)MT-IIA isoform is induced in carcinoma cells (A431, SiHa, and HT29) exposed to low oxygen, conditions commonly found in solid tumors. The present study demonstrates that the genes for hMT-IIA and mouse (m)MT-I are transcriptionally activated by hypoxia through metal response elements (MREs) in their proximal promoter regions. These elements bind metal transcription factor-1 (MTF-1). Deletion and mutational analyses of the hMT-IIA promoter indicated that the hMRE-a element is essential for basal promoter activity and for induction by hypoxia, but that other elements contribute to the full transcriptional response. Functional studies of the mMT-I promoter demonstrated that at least two other MREs (mMRE-d and mMRE-c) are responsive to hypoxia. Multiple copies of either hMRE-a or mMRE-d conferred hypoxia responsiveness to a minimal MT promoter. Mouse MT-I gene transcripts in fibroblasts with targeted deletions of both MTF-1 alleles (MTF-1(-/-); dko7 cells) were not induced by zinc and showed low responsiveness to hypoxia. A transiently transfected MT promoter was unresponsive to hypoxia or zinc in dko7 cells, but inductions were restored by cotransfecting a mouse MTF-1 expression vector. Electrophoretic mobility shift assays detected a specific protein-DNA complex containing MTF-1 in nuclear extracts from hypoxic cells. Together, these results demonstrate that hypoxia activates MT gene expression through MREs and that this activation involves MTF-1.

Regulation of expression of c-fos and c-myc in rat lymphoma Nb-2 cells.

This study examined the effects of the mitogen, prolactin and the cell cycle inhibitors, cyclosporin A and neomycin sulfate, on expression of the proto-oncogenes c-fos and c-myc in the rat lymphoma Nb-2 cell line. Stimulation of quiescent cultures with prolactin resulted in a 2-3-fold increase in the constitutive levels of c-myc mRNA which peaked at 4 h and declined thereafter. c-Fos mRNA was not detected in quiescent or prolactin-stimulated cultures. Cyclosporin A or neomycin sulfate reversibly blocked the mitogenic effect of prolactin on Nb-2 cells, but had little effect on constitutive levels of c-myc. However, the release of Nb-2 cells from a cyclosporin A or a neomycin sulfate block resulted in a rapid transient induction of c-fos which peaked at 0.5-1 h and declined rapidly thereafter. These results indicate that the rapid transient expression of c-fos following release from cell cycle blockage was not sufficient to elicit cell division, but these cells were competent to respond to prolactin. Prolactin allows progression through the cell cycle and enhances c-myc mRNA levels.

Metallothionein mRNA stability in chicken and mouse cells.

Northern blot analysis revealed that metallothionein (MT) mRNAs accumulate after inhibition of protein synthesis with cycloheximide (CHX) in primary cultures of chick embryo hepatocytes and fibroblasts, as well as in an established mouse hepatoma cell line. Inhibition of RNA synthesis with actinomycin D (AMD) led to rapid loss of MT mRNAs in these cells, whereas CHX dramatically retarded the rate of MT mRNA decay (t1/2 greater than 24 h). These results suggest that CHX causes MT mRNA accumulation primarily by increasing stability of MT mRNA. Thus, changes in MT mRNA turn-over rates may play an important role in regulating the accumulation of MT mRNA. The half-lives of MT mRNAs in chicken and mouse cells were determined by oligodeoxyribonucleotide excess solution hybridization with RNA samples extracted after different periods of exposure to AMD. The half-life of chicken MT (cMT) mRNA in uninduced chicken embryo hepatocytes was 3.6 h. Induction of cMT mRNA by pretreatment of these cells with zinc (Zn) prior to exposure to AMD, did not alter the half-life of cMT mRNA significantly. In contrast, cadmium (Cd) induction led to a 2.5-fold increase in the stability of this mRNA. In uninduced chicken embryo fibroblasts, cMT mRNA levels were too low to allow accurate determination of half-life using the methods employed here. However, the half-life of this mRNA in Zn-induced chicken embryo fibroblasts was 6.2 h, whereas it was 9.3 h in Cd-induced cells. Thus, the turn-over rate of cMT mRNA after Cd-induction is very similar in chick embryo fibroblasts and hepatocytes. These data suggest that the accumulation of MT mRNA in chicken cells may reflect, in part, metal-specific effects on MT mRNA stability. The half-lives of mouse MT-I and MT-II (mMT-I and mMT-II) mRNAs in uninduced BNL hepatoma cells were identical (9.2 h), and were not effectively altered after induction by metals (Zn, Cd) or interleukin-1 beta (IL-1 beta). However, mMT mRNAs in pachytene spermatocytes and round spermatids, freshly isolated from the adult testes, were 2.2- to 4.5-fold more stable than in hepatoma cells. These results suggest that cell-type specific accumulation of mMT mRNAs may be regulated, in part, by mRNA stability.

High levels of metallothionein messenger RNAs in male germ cells of the adult mouse.

Northern blotting, in situ hybridization, and oligodeoxyribonucleotide excess solution hybridization were used to quantitate metallothionein-I (MT-I) and MT-II mRNAs in mouse testes. Testes from sexually mature adults contained high levels of both MT mRNAs (approximately 10-fold higher than those in control adult liver). Testicular MT mRNA levels were age dependent, being low the first 2 weeks after birth and increasing slowly thereafter to maximal levels in the adult (by 9 weeks after birth). In the adult testis, in situ hybridization indicated that only cells within the adluminal compartment (germ cells) of the seminiferous tubules contain high levels of MT mRNA. The appearance of cells containing elevated levels of MT mRNA during development was delayed from the onset of spermatogenesis. In situ hybridization suggested that MT mRNA accumulates after the initial differentiation of primary spermatocytes and is maintained in spermatids. Pachytene spermatocytes (PSC) and round spermatids (RTD) isolated from adult testes contained both MT-I and MT-II mRNAs in levels equivalent to those found in zinc-treated hepatocytes, whereas very low levels of MT mRNA were detected in isolated Sertoli cells (ST). In situ hybridization suggested that MT mRNA was present at only basal levels in interstitial, spermatogonial, and mature sperm cells at all developmental stages examined. Northern blot and in situ hybridization to sulfated glycoprotein-2 (SGP-2) mRNA, a ST-specific transcript, showed that SGP-2 mRNA is high in the testis of 1-week-old mice and decreases gradually to a lower level in the adult. In situ detection of this mRNA was consistent with the location of ST in the testis. SGP-2 mRNA was abundant in ST and rare in PSC and RTD preparations. Analysis of pulse-labeled proteins from isolated PSC and RTD indicated that these cells actively synthesize MT-I and MT-II. The high levels of MT mRNA in adult testes were not increased substantially after systemic injection of cadmium, zinc, or bacterial lipopolysaccharide. In marked contrast, these treatments led to dramatically increased levels of hepatic and ovarian MT mRNA. This study establishes that the MT genes are actively expressed in a developmentally regulated fashion in the male germ cells of the mouse. This suggests a role for MT in the process of spermatogenesis.

Lactoferrin in the mouse uterus: analyses of the preimplantation period and regulation by ovarian steroids.

Uterine expression of lactoferrin (LF) during the preimplantation period and its regulation by the ovarian steroids estradiol (E2) and/or progesterone (P4) in ovariectomized adult mice were examined. Immunoblot detection of LF in uterine cell lysates demonstrated the presence of this protein from days 1-8 of pregnancy [day 1 (D1) = day of vaginal plug]. Immunoprecipitation of 35S pulse-labeled uterine proteins showed that the relative rate of LF synthesis was high on D1, but below the level of detection by D4. Immunolocalization of LF in uterine sections showed intense luminal and glandular epithelial staining on D1 and D2, and progressively decreased staining through D4. Immunoreactive protein was also detected in cells, primarily concentrated in the stroma. The relative number of these cells was greatest on D1 and decreased progressively to a low number by D4. These cells were morphologically similar to neutrophils, which are known to contain LF protein, but little or no LF mRNA. Northern blotting showed that uterine LF mRNA levels were very high on D1 and D2 of pregnancy and decreased to low, but detectable, levels by D4. In situ hybridization to uterine sections showed that LF mRNA was highly abundant only in glandular and luminal epithelial cells, and followed the same pattern as immunolocalization on D1-D4 in epithelial cells. These results document two sources of LF in the preimplantation mouse uterus: neutrophils and epithelial cells. The synthesis of LF in the uterus reflects the abundance of epithelial LF mRNA, which is high on the first 2 days of pregnancy. Neutrophils that contain LF are also abundant in the uterine stroma during this time. E2 and/or P4 regulation of uterine LF was examined. LF mRNA was rapidly induced by E2 in ovariectomized adult mice, and this mRNA was localized exclusively to epithelial cells. P4 had little effect on uterine LF mRNA levels, but antagonized the prolonged induction of this gene by E2. E2 induced the accumulation of immunoreactive LF in uterine epithelial cells and the appearance of numerous immunopositive neutrophils distributed throughout the uterine stroma. P4 also antagonized these effects. Thus, E2 regulates LF gene expression in uterine epithelial cells and causes the recruitment of neutrophils into the uterus. These results suggest that LF may play an important role in early pregnancy and that uterine LF gene expression is regulated by a balance between estrogen and P4.

Cell type-specific expression of transforming growth factor-beta 1 in the mouse uterus during the periimplantation period.

Immunohistochemistry and in situ and Northern blot hybridization were employed to determine temporal and spatial expression of transforming growth factor-beta 1 (TGF beta 1) in the mouse uterus during the periimplantation period. The polyclonal antisera anti-LC-(1-30) and anti-CC-(1-30), raised against two different preparations of a peptide corresponding to the amino-terminal 30 amino acids of TGF beta 1, were used for histochemical analyses because of their distinct staining patterns. Anti-LC shows intracellular staining, while staining by anti-CC is primarily extracellular. The colocalization of intracellular staining by anti-LC with in situ hybridization of TGF beta 1 mRNA in the luminal and glandular epithelia on days 1-4 of pregnancy (day 1 = vaginal plug) indicates that the epithelial cells are the primary sites of TGF beta 1 synthesis during the preimplantation period. On the other hand, staining of the extracellular matrix of the stroma by anti-CC during this period suggests an active accumulation of TGF-beta 1 that is synthesized in and secreted from the epithelia. While intracellular staining and accumulation of TGF-beta 1 mRNA in the epithelia were clearly evident on days 1-4, the extracellular staining showed temporal fluctuations. The clear extracellular staining of the stroma that was observed on day 1 was absent on day 2; moderate staining was again visualized in the stroma on day 3 and was markedly increased on day 4.(ABSTRACT TRUNCATED AT 250 WORDS)

Estrogen regulates the synthesis of epidermal growth factor in mouse uterine epithelial cells.

Immunocytochemical analyses, using several mouse epidermal growth factor (EGF) polyclonal antibodies, detected immunoreactivity only in uterine luminal and glandular epithelia on late proestrus, estrus, and early on day 1 of pregnancy, but not late on day 1. This immunoreactivity was not detected in the ovariectomized uterus, but after estrogen stimulation it was detected first in the luminal epithelium between 12-24 h and then also in the glandular epithelium by 48 h. After 72 h of estrogen withdrawal, EGF immunoreactivity was no longer detected. This response was specific for estrogen and did not occur after progesterone injection (2 mg/day for 4 days). Using antipeptide antibodies specific for prepro-EGF, no immunoreactivity was detected in the ovariectomized uterus, weak reactivity was detected in the estrogenized uterus and submandibular gland, and strong reactivity was detected in the kidney. Northern blot analysis of uterine RNA failed to detect the expected 4.8-kilobase prepro-EGF mRNA, but, instead, a rare transcript of 2.4 kilobases was detected, which suggests that EGF mRNA is alternately processed in the uterus. The presence of an EGF-coding uterine transcript was further documented by hybridization of an EGF-coding region-specific oligodeoxyribonucleotide (oligo) to polymerase chain reaction-amplified uterine cDNA. In situ hybridization, using a prepro-EGF cRNA probe as well as an EGF-coding region-specific oligo, showed hybridization that colocalized with the EGF immunostaining (epithelia) and was absent from non-EGF-immunoreactive cells. Pulse labeling experiments coupled with immunoaffinity chromatography showed that estrogen induced an increase in the relative rate of synthesis of an acid-soluble immunoreactive protein which was the same size as authentic EGF. Furthermore, analysis of acid-soluble uterine proteins fractionated by DEAE-cellulose chromatography demonstrated a single coincident peak of antigenic activity and receptor-binding activity which coeluted from the column with authentic EGF. Electron microscopy localized EGF immunoreactivity to the Golgi of luminal epithelial cells. Taken together these results suggest that estrogen regulates expression of the EGF gene specifically in uterine epithelial cells. Increased expression of this gene results in an increase in the relative rate of synthesis of this protein and the accumulation of mature EGF.

Cell type-specific localization of c-myc protein in the mouse uterus: modulation by steroid hormones and analysis of the periimplantation period.

Estrogen has been shown to induce a rapid transient increase in c-myc mRNA in the rat uterus. However, no studies of the cell specificity of c-myc expression in the uterus have been reported, and nothing is known about the expression of c-myc in response to other steroids or during normal uterine preparation for implantation. To this end, the cell type-specific localization of c-myc protein was determined in the ovariectomized mouse uterus after progesterone (P4) and/or 17 beta-estradiol (E2) injection as well as during the periimplantation period. After E2 injection, a rapid accumulation of c-myc protein was detected exclusively in the uterine luminal and glandular epithelial nuclei in the ovariectomized mouse. Essentially all of these cells contained immunoreactive c-myc by 12 h postinjection. In contrast, after P4 injection, rapid accumulation of c-myc was noted exclusively in some of the stromal cell nuclei. Pretreatment of the ovariectomized mouse for 4 days with P4 (P4 priming) followed by E2 injection resulted in an increase in the number of c-myc-positive stromal cells, but few, if any, c-myc-positive cells were detected in the luminal and glandular epithelia. These uterine cell type-specific localizations of c-myc protein, induced by E2 or P4 injection, were followed within 18-24 h by DNA synthesis ([ 3H]thymidine incorporation) restricted to the epithelia or stroma, respectively. c-Myc was detected in the nuclei of luminal and glandular epithelia during proestrus and on days 1 and 2 of pregnancy, a period when the uterus is under the influence of estrogen. c-Myc-positive cells were detected in the stroma on day 3, and by day 4 a large number of stromal cell nuclei were c-myc positive. These changes are coincident with increasing P4 levels during early pregnancy. At the implantation chamber on day 5, many cells in the primary decidual zone as well as some of the deeper stromal cells were c-myc protein positive, whereas on day 6, c-myc protein was detected only in the secondary decidual zone. During this period of uterine preparation for embryo implantation and subsequent decidualization, there was a positive correlation between c-myc protein localized in specific populations of uterine cells and subsequent DNA synthesis in those cell types. Thus, both E2 and P4 induce cell type-specific accumulation of c-myc protein in the uterus of the ovariectomized mouse, with E2 induction of c-myc being restricted to epithelia, and P4 induction restricted to stroma.(ABSTRACT TRUNCATED AT 400 WORDS)

Differential expression and regulation of cytokine genes in the mouse uterus.

The present investigation examined the differential expression of cytokine genes in vivo and in vitro in the mouse uterus and their regulation by ovarian steroid hormones. The expression of interleukin-1 beta (IL-1 beta), IL-1 alpha, IL-6, and tumor necrosis factor-alpha (TNF alpha) genes was examined in the mouse uterus as well as in freshly isolated or cultured epithelial cells by Northern blot and in situ hybridization. In the day 1 pregnant (D1 = vaginal plug) uterus, the levels of IL-1 alpha and IL-1 beta messenger RNAs (mRNAs) were abundant, whereas those of TNF alpha and IL-6 were at the limits of detection. Freshly isolated D1 uterine epithelial cell preparations contained higher levels of IL-1 alpha and IL-1 beta mRNAs than those observed in the D1 whole uterus, whereas TNF alpha and IL-6 mRNA levels were consistently low. In contrast, D1 epithelial cells showed decreased levels of IL-1 beta mRNA after 1 day of culture, whereas the levels of IL-1 alpha and IL-6 mRNAs increased under similar conditions. The levels of IL-1, IL-6, and TNF alpha mRNAs were at the limits of detection in the D4 whole uterus or freshly isolated diestrous epithelial cells. However, IL-1 alpha and IL-6 mRNA levels in diestrous epithelial cells, like those in D1 epithelial cells, increased in culture. In contrast, IL-1 beta and TNF alpha mRNA levels remained low in cultured epithelial cells. In situ hybridization was used to examine the cell type-specific expression of IL-1 alpha or IL-6 mRNA in uterine sections and cultured cells. Although hybridization signals for IL-1 alpha mRNA were detected in uterine epithelial cells on D1 of pregnancy, IL-6 mRNA could not be detected. IL-1 alpha and IL-6 mRNAs could not be detected in freshly isolated diestrous epithelial cells, although a majority of the epithelial cells showed hybridization signals for these mRNAs after 2 or 4 days of culture. The effects of steroid hormones on uterine cytokine gene expression were examined by Northern blot and in situ hybridization. In adult ovariectomized mice, an injection of 17 beta-estradiol (E2), progesterone (P4), or a combination of E2 and P4 had little or no apparent effect on these cytokine mRNA levels. The results establish that uterine epithelial cells on D1 of pregnancy exhibit heightened expression of IL-1 alpha in culture. In contrast, these cells express little or no IL-6 mRNA in vivo, but show heightened expression in culture. These results suggest that an apparent loss of repression of these uterine genes occurs in culture. Furthermore, E2 and/or P4 treatments appear to have little or no effect on uterine cytokine mRNA levels in adult ovariectomized mice.

Regulation of avian calbindin-D28K gene expression in primary chick kidney cells: importance of posttranscriptional mechanisms and calcium ion concentration.

Vitamin D-dependent calcium-binding protein (calbindin-D28K), is regulated by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], and several other factors in a tissue-specific manner, but the controlling mechanisms are still poorly understood. In this study we examined the relative contributions of transcriptional and posttranscriptional mechanisms in the 1,25-(OH)2D3 control of calbindin-D28K mRNA expression in primary chick kidney cells and investigated the effect of extracellular Ca2+ on calbindin-D28K gene expression in the presence and absence of hormone. 1,25-(OH)2D3 treatment (10(-8) M) of cells grown in serum-free medium resulted in a marked 20- to 30-fold increase in calbindin-D28K mRNA peaking at 12-18 h, which then rapidly declined to basal levels by 24 h. The abrupt decline in mRNA appeared to be associated with a reduction in size of the calbindin-D28K transcripts. Nuclear run-off assays showed only a slight (1.5-fold) increase in calbindin-D28K gene transcription 2 h after 1,25-(OH)2D3, whereas parallel assays clearly demonstrated a marked (7-fold) induction in the rate of metallothionein gene transcription 2 h after treatment of chick kidney cells with 10 microM zinc. The induction of calbindin-D mRNA by 1,25-(OH)2D3 required ongoing protein synthesis, since it was blocked by cycloheximide. Calbindin-D28K mRNA was stable for 12 h in the presence of actinomycin-D in both vitamin D-deficient and 1,25-(OH)2D3-treated cells. Both basal and 1,25-(OH)2D3-induced calbindin-D28K mRNA were modulated by the extracellular Ca2+, with maximum expression occurring at 1-2 mM. We conclude that 1,25-(OH)2D3 induces kidney calbindin-D28K mRNA by producing a small increase in its transcriptional rate, which is accompanied by pronounced posttranscriptional effects(s). The striking modulation of calbindin-D28K expression by extracellular Ca2+ is consistent with a putative role for this protein in the regulation of this ion in the kidney cell.

Expression of transforming growth factor-beta isoforms (beta 2 and beta 3) in the mouse uterus: analysis of the periimplantation period and effects of ovarian steroids.

Expression of beta-type transforming growth factor genes (TGF beta 2 and TGF beta 3) in the mouse uterus during the periimplantation period and in response to an acute exposure to 17 beta-estradiol (E2) and progesterone (P4) was studied using Northern blot hybridization and/or immunocytochemistry. Polyclonal antipeptide antibodies specific for TGF beta 2 or TGF beta 3 were employed for immunocytochemistry. In the preimplantation uterus [days (D) 1-4 of pregnancy; day 1 = vaginal plug], immunostaining for TGF beta 2 was observed in luminal and glandular epithelia as well as in myometrium and vascular smooth muscle. In the postimplantation period (D5-D8), TGF beta 2 immunostaining was also detected in decidual cells. In contrast, TGF beta 3 immunostaining was restricted to the myometrium and vascular smooth muscle throughout the periimplantation period (D1-D8). Antisense TGF beta 2 and TGF beta 3 RNA probes were employed for Northern blotting. Northern blot hybridization revealed four TGF beta 2 transcripts (approximately 6.0, 5.0, 4.0, and 3.5 kilobases) in total uterine poly(A)+ RNA on D1-D6 and in poly(A)+ RNA from the deciduum and myometrium collected on D7 and D8 of pregnancy. These TGF beta 2 transcripts were also detected in isolated samples of deciduomata or myometrium obtained from D8 pseudopregnant mice in which the decidual cell reaction was induced experimentally on D4. The levels of these transcripts remained relatively constant during the periimplantation period. Northern blot analysis detected a 3.8-kilobase TGF beta 3 transcript in total uterine poly(A)+ RNA on D1-D6. This transcript was detected in myometrial RNA samples on D7 and D8 of pregnancy or D8 of pseudopregnancy, but was not detected in RNA from the deciduum on D7 and D8 or in that from deciduomata on D8. The effects of ovarian steroids on TGF beta 2 and TGF beta 3 mRNAs were examined in uteri of adult ovariectomized mice. Uterine TGF beta 2 or TGF beta 3 mRNA persisted in ovariectomized mice. However, an injection of E2 induced a rapid (6 h), but transient, induction (approximately 3- to 4-fold) of TGF beta 2 mRNA. An injection of P4 had no effect on TGF beta 2 mRNA levels, and coinjection of P4 with E2 did not antagonize the E2-stimulated transient accumulation of TGF beta 2 mRNA. In comparison, neither an injection of E2 nor one of P4 exerted significant effects on TGF beta 3 mRNA levels.(ABSTRACT TRUNCATED AT 400 WORDS)

Differential expression of epidermal growth factor receptor (EGF-R) gene and regulation of EGF-R bioactivity by progesterone and estrogen in the adult mouse uterus.

The present study examined several aspects of epidermal growth factor receptor (EGF-R) in the mouse uterus during the peri-implantation period and after ovarian hormone treatment of adult ovariectomized mice. The cell-specific distribution, regulation of expression, and binding kinetics were assessed by immunohistochemistry, Northern blot analysis, and ligand binding assays, respectively. Affinity cross-linking studies ascertained the size of the EGF-R, and its bioactivity was examined by determining EGF-dependent subcellular protein tyrosine kinase activity and receptor autophosphorylation. In the intact uterus and separated cell types, EGF-R was detected in the stroma, deciduum, and myometrium, but not in the luminal or glandular epithelium. Uterine EGF-R mRNA transcript profiles showed some differences between pregnant and ovariectomized mice regardless of steroid hormone treatments. Two major [6.5- and 2.7-kilobase (kb)] and two less abundant (9.6- and 5.0-kb) transcripts were detected in pregnant uterine poly(A)+ RNA. Three additional transcripts (< 2.0 kb) were detected in decidual poly(A)+ RNA, and a larger transcript (8.0 kb) was detected in uterine poly(A)+ RNA isolated from ovariectomized mice. Scatchard analysis of EGF binding also revealed apparent differences in binding kinetics between pregnant and ovariectomized mice, although EGF was cross-linked to a 170-kilodalton protein under these conditions. Two classes (Kd, approximately 0.2 and approximately 2.0 nM) of binding sites were noted in pregnant mice, whereas a single class (Kd, approximately 1.0 nM) was found in ovariectomized mice. 17 beta-Estradiol (E2) caused a rapid transient upregulation of uterine EGF-R mRNA levels and increased the number of EGF-binding sites in ovariectomized mice, as did an injection of progesterone (P4). However, the bioactivity of EGF-R could not be detected in uteri of ovariectomized mice treated with oil or P4. E2 treatment was found to be essential for EGF-R bioactivity. Taken together, the results suggest that in the adult mouse uterus, EGF-R status is influenced by factors other than P4 and E2, the epithelium is not the direct target for the actions of EGF-related growth factors as thought previously, the mitogenic effects of these growth factors on epithelial cells in vivo are perhaps mediated by other uterine cell-types expressing EGF-R, and, lastly, these growth factors are not likely to be functional in the uterus in the absence of estrogen. The present observations are supportive of the concept of paracrine or juxtacrine interactions between EGF-related growth factor ligands of luminal epithelial origin and blastocyst EGF-R in the process of implantation.

Cloning and expression of an avian metallothionein-encoding gene.

The chicken metallothionein gene (cMT), isolated from a chicken genomic DNA phage lambda library, was found to be approximately 1.5 kb in length and to consist of three exons, separated by two intervening sequences. The number and placement of the introns in the cMT gene is precisely the same as that in the mammalian metallothionein-coding genes. S1 nuclease mapping indicated a prominent transcription start point (tsp) 62 bp 5' to the translation start codon. The promoter region analyzed (623 bp) contained three regions of homology (at -47, -488, and -577 bp relative to the tsp) with the metal regulatory element (MRE) consensus sequence, and three potential Sp1 binding sites. Two of the putative MREs (-47, -577) were 12 to 14-bp palindromes, which suggests that they are binding sites for trans-acting proteins. The intact cMT gene was functional in mammalian cells, and the cMT promoter could confer metal responsiveness on the firefly luciferase cDNA (Luc) in transient expression assays. Deletion mutagenesis established that 107 bp of 5'-flanking sequence, containing the proximal MRE and a putative Sp1-binding site, were sufficient for transient expression and metal induction of the cMT promoter-Luc fusion gene.

Regulation of metallothionein gene expression by oxidative stress and metal ions.

The metallothioneins (MT) are small, cysteine-rich heavy metal-binding proteins which participate in an array of protective stress responses. Although a single essential function of MT has not been demonstrated, MT of higher eukaryotes evolved as a mechanism to regulate zinc levels and distribution within cells and organisms. These proteins can also protect against some toxic metals and oxidative stress-inducing agents. In mice, among the four known MT genes, the MT-I and -II genes are most widely expressed. Transcription of these genes is rapidly and dramatically up-regulated in response to zinc and cadmium, as well as in response to agents which cause oxidative stress and/or inflammation. The six zinc-finger metal-responsive transcription factor MTF-1 plays a central role in transcriptional activation of the MT-I gene in response to metals and oxidative stress. Mutation of the MTF-1 gene abolishes these responses, and MTF-1 is induced to bind to the metal response elements in proximal MT promoter in cells treated with zinc or during oxidative stress. The exact molecular mechanisms of action of MTF-1 are not fully understood. Our studies suggest that the DNA-binding activity of MTF-1 in vivo and in vitro is reversibly activated by zinc interactions with the zinc-finger domain. This reflects heterogeneity in the structure and function of the six zinc fingers. We hypothesize that MTF-1 functions as a sensor of free zinc pools in the cell. Changes in free zinc may occur in response to chemically diverse inducers. MTF-1 also exerts effects on MT-I gene transcription which are independent of a large increase in MTF-1 DNA-binding activity. For example, cadmium, which has little effect on the DNA-binding activity of MTF-1 in vivo or in vitro, is a more potent inducer of MT gene expression than is zinc. The basic helix-loop-helix-leucine zipper protein, USF (upstream stimulatory factor family), also plays a role in regulating transcription of the mouse MT-I gene in response to cadmium or H2O2. Expression of dominant negative USF-1 or deletion of its binding site from the proximal promoter attenuates induction of the mouse MT-I gene. USF apparently functions in this context by interacting with as yet unidentified proteins which bind to an antioxidant response element which overlaps the USF-binding site (USF/ARE). Interestingly, this composite element does not participate in the induction of MT-I gene transcription by zinc or redox-cycling quinones. Thus, regulation of the mouse MT-I gene by metals and oxidative stress involves multiple signaling pathways which depend on the species of metal ion and the nature of the oxidative stress.

Competition between iron(III)-selective chelators and zinc-finger domains for zinc(II).

Many iron(III)-selective chelators possess an appreciable affinity for zinc(II) and this can prove to be undesirable when such chelators are being assessed for clinical application. At present, there is no useful test available which can reliably access this problem. In the present manuscript, we provide evidence that indicates that a zinc-finger protein MTF-1, (metal transcription factor-1) may prove to be a suitable candidate. N,N',2-hydroxybenzyl ethylenediamine diacetic acid, in contrast to desferrioxamine, removes zinc quite efficiently from MTF-1.

Analysis of the effects of overexpression of metallothionein-I in transgenic mice on the reproductive toxicology of cadmium.

Exposure to low levels of cadmium reduces fertility. In male mice spermatogenesis is highly sensitive to cadmium, whereas in females the peri-implantation period of pregnancy is sensitive. To examine the potential roles of the cadmium-binding protein, metallothionein (MT), in the reproductive toxicology of cadmium, we examined a transgenic mouse strain that overexpresses metallothionein-I (MT-I). These mice had dramatically increased steady-state levels of MT-I mRNA and MT in the testes and in the female reproductive tract during the peri-implantation period of pregnancy, and this overexpression occurred in a cell-specific and temporally regulated manner similar to that of the endogenous MT-I gene. Transgenic and control males were injected with cadmium, and the histology of the testes was examined. An injection of 7.5 mumol Cd/kg had no effect on histology of the testes in either transgenic or control mice. In contrast, an injection of 10 mumol Cd/kg caused rapid changes in the histology of the testes and resulted in pronounced testicular necrosis in both control and transgenic mice. Female transgenic and control mice were mated and then injected with cadmium (30-45 mumol Cd/kg) on the day of blastocyst implantation (day 4). In both of these groups, injection of cadmium reduced pregnancy rate, and no dramatic protection was afforded by maternal and/or embryonic overexpression of MT. Thus, overexpression of MT-I does not significantly protect against either of these cadmium-induced effects on fertility.

Regulation of interleukin-6 and interleukin-1 beta gene expression in the mouse deciduum.

Expression and regulation of interleukin-6 (IL-6) and IL-1 beta were examined in the mouse deciduum and in experimentally induced deciduoma from 6 to 8 days postcoitum (1 dpc = vaginal plug), as well as in cultured mouse decidual cell preparations. Levels of these mRNAs in the deciduum and deciduoma were below the limits of detection by Northern blotting. However, enzymatic dispersion and culture of decidual cells and/or exposure to bacterial endotoxin-lipopolysaccharide (LPS) induced these mRNAs. IL-6 levels that accumulated in the culture medium (3990 pg/3 x 10(6) cells/day) were about 90-times higher than those of IL-1 beta (45 pg/3 x 10(6) cells/day). Progesterone (10(-7) M) modestly (40%) reduced the levels of IL-6 mRNA and protein during culture, whereas LPS dramatically (8-fold) and rapidly induced IL-6 and IL-1 beta mRNAs and proteins. In vivo, few IL-1 beta immunopositive cells were localized by immunohistochemistry in the 8 dpc deciduum. In contrast, IL-6 mRNA was localized by in situ hybridization in dispersed clusters of a few cells in the mesometrial deciduum near the center of the implantation site. LPS rapidly induced interleukin mRNAs in the deciduum and deciduoma. After LPS injection, IL-1 beta immunopositive cells were dispersed in the myometrium and mesometrial deciduum. In contrast, after LPS injection (2 h), IL-6 mRNA was abundant in 'cords' of cells that traverse the mesometrial deciduum longitudinally, as well as in cells dispersed throughout the myometrium. Thus, the IL-1 beta and IL-6 genes are expressed and regulated in distinct subsets of cells in the decidual bed. The pattern of F4/80 immunostaining is consistent with macrophages as the major, if not only, source of decidual IL-1 beta. IL-6 is also expressed in these cells. However, IL-6 gene expression is regulated in a distinct subset of cells located in the mesometrial decidual bed of the mouse.

Effects of 9-ene-tetrahydrocannabinol on expression of beta-type transforming growth factors, insulin-like growth factor-I and c-myc genes in the mouse uterus.

Effects of cannabinoid on expression of beta-type transforming growth factors (TGF-beta 1, -beta 2 and -beta 3), insulin-like growth factor-I (IGF-I) and c-myc genes in the uteri of adult ovariectomized mice were examined using Northern blot hybridization. Mice were exposed to 9-ene-tetrahydrocannabinol (THC) alone or in combination with an injection of estradiol-17 beta (E2) and/or progesterone (P4), and uteri were analyzed at various times thereafter. TGF-beta isoform messenger RNAs (mRNAs) persisted in ovariectomized uteri and their levels were not altered after THC treatment, whereas an injection of E2 caused a modest increase in TGF-beta 1 and -beta 3 mRNA levels at 24 h. Imposition of THC treatment advanced the stimulatory effects of E2 by changing the timing for the peak of TGF-beta 3 mRNA levels to 12 h. In comparison, E2 treatment substantially elevated the levels of TGF-beta 2 mRNA at 6 h, and THC potentiated this E2 response without affecting the timing for the response. Imposition of P4 treatment did not antagonize any of these responses. P4 treatment alone or with THC had insignificant effects on mRNA levels for these TGF-beta isoforms. Uterine levels of IGF-I and c-myc mRNAs were low in ovariectomized mice and THC did not alter these mRNA levels. In contrast, E2 treatment induced a rapid, but transient, increase in IGF-I and c-myc mRNAs, and THC antagonized the rapid c-myc mRNA response and altered the timing of the IGF-I mRNA response. P4 treatment alone also caused the transient induction of these mRNAs, but THC failed to antagonize these effects. An injection of P4 plus E2 resulted in further modest increases in IGF-I and c-myc mRNA levels as compared to E2 or P4 treatment alone. However, THC did not antagonize these transient stimulatory effects of the combined ovarian steroids. The data suggest that THC should not be classified as estrogenic or antiestrogenic. However, this compound can modulate (potentiate, antagonize and/or alter timing) the effects of ovarian steroids on uterine gene expression.