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W L Zheng

Publications and source records attributed to W L Zheng.

13 recordsLinked to original sources
PubMed2001

Retinoic acid biosynthesis by normal human breast epithelium is via aldehyde dehydrogenase 6, absent in MCF-7 cells.

Retinoic acid (RA) is the form of vitamin A that controls differentiation and proliferation of epithelia. Our previous work established that normal breast epithelia synthesize RA from retinol, an ability retained by three immortalized but nontumorigenic cell lines but lost in five of six breast cell lines. In this work, we characterize the cause of this defect in one of the lines, the MCF-7 line. We have determined that the immortalized but nontumorigenic cell line, MTSV1.7, capable of synthesizing RA from both retinol and retinal, contains a retinaldehyde dehydrogenase activity for the second step in RA biosynthesis. We have identified it, after isolation, as a previously described enzyme, aldehyde dehydrogenase 6 (ALDH6). Immunohistochemical analysis of normal human breast with antibodies to ALDH6 showed expression of this enzyme in the glandular epithelia colocalized with cellular RA-binding protein type II, a possible marker for certain cells able to synthesize RA. ALDH6 was not present in MCF-7 cells, and these cells were unable to oxidize retinal to RA in culture. When MCF-7 cells were then transfected with ALDH6, they (re)gained the ability to oxidize retinal to RA as well as some ability to synthesize RA when provided with retinol. This suggests that loss of ALDH6 expression is the defect in RA biosynthesis in these cells. Identification of ALDH6 as the retinaldehyde dehydrogenase present in normal human breast epithelia provides the first tool necessary for studying the loss of RA synthetic ability in cancer cells and the relationship of this process to malignant transformation.

Aldehyde Dehydrogenase↗
PubMed2000

Retinol conversion to retinoic acid is impaired in breast cancer cell lines relative to normal cells.

The bioactivity of retinol (vitamin A) is in part dependent on its metabolism to retinoic acid (RA). We investigated the ability of breast epithelial cells to synthesize RA when challenged with a physiological retinol dose (2 microM). Normal human mammary epithelial cells (HMEC) cultured from reduction mammoplasties were competent in RA synthesis and the ability to synthesize RA was retained by immortal, nontumorigenic breast epithelial cell lines (MTSV1.7, MCF-10F, and 184B5). In contrast, most (five of six) breast cancer cell lines could not synthesize RA or did so at low rates relative to normal cells. A notable exception was the MDA-MB-468 cell line, which was fully competent in RA synthesis. Most (>/=68%) of the RA synthesized by breast cells was recovered from the culture medium. Cellular retinol binding protein and cellular RA binding protein II, both expressed in HMEC, had various expression patterns in the cell lines that did not correlate with the observed differences in RA synthesizing ability. Strong RA induction of the RA hydroxylase P450RAI (CYP26) was confined to ERalpha-positive T47D and MCF-7 breast cancer cells and did not appear to explain the lack of detectable RA levels in these cells since RA remained undetectable when the cells were treated with 5-10 microM liarozole, a P450RAI inhibitor. We hypothesize that retinol bioactivity is impaired in breast cancer cells that cannot synthesize RA. In preliminary support of this hypothesis, we found that retinol (0.5-2 microM) inhibited MCF-10F but not T47D or MCF-7 cell growth.

Breast↗
PubMed2000

Retinoic acid synthesis and expression of cellular retinol-binding protein and cellular retinoic acid-binding protein type II are concurrent with decidualization of rat uterine stromal cells.

Decidualization of stromal cells at the site of embryo implantation in the rat uterus is accompanied by expression of cellular retinol-binding protein and cellular retinoic acid-binding protein [CRABP(II)], whose presence has been shown to correlate with gain of ability to synthesize retinoic acid in other cells. Here we examined whether decidual cells also acquired the ability to synthesize retinoic acid, which would have important implications for understanding the implantation process. Decidual cells were isolated from the uterus on day 8 of pregnancy and cultured. When provided with retinol, they indeed synthesized and released retinoic acid to the medium. To follow acquisition of this ability more closely, artificial induction of decidualization was exploited. Ovariectomized rats were placed on a hormonal regimen that allows decidualization to occur in vivo, with oil stimulation, or in vitro, if cells are isolated on day 5 of the regimen and then cultured. Decidualization in vivo reproduced the expression of cellular retinol-binding protein and CRABP(II) seen during pregnancy. Stromal cells isolated on regimen day 2 synthesized little retinoic acid and expressed little alkaline phosphatase, a marker of decidualization. Stromal cells isolated on regimen day 5 had elevated levels of alkaline phosphatase, increasing during the 3 days of culture examined. The ability of the stromal cells to synthesize retinoic acid showed the same pattern: a substantially elevated production from that previously observed, on day 2, with production increasing significantly over the next 2 culture days. Thus, expression of CRABP(II) was correlated with gain of ability to synthesize retinoic acid. Retinoid signaling may be an important part of the process of embryo implantation.

Alkaline Phosphatase↗
PubMed1999

A 5-kilobase pair promoter fragment of the murine epididymal retinoic acid-binding protein gene drives the tissue-specific, cell-specific, and androgen-regulated expression of a foreign gene in the epididymis of transgenic mice.

The murine epididymis synthesizes and secretes a retinoic acid-binding protein (mE-RABP) that belongs to the lipocalin superfamily. The gene encoding mE-RABP is specifically expressed in the mouse mid/distal caput epididymidis under androgen control. In transgenic mice, a 5-kilobase pair (kb) promoter fragment, but not a 0.6-kb fragment, of the mE-RABP gene driving the chloramphenicol acetyltransferase (CAT) reporter gene restricted high level of transgene expression to the caput epididymidis. No transgene expression was detected in any other male or female tissues. Immunolocalization of the CAT protein and in situ hybridization of the corresponding CAT mRNA indicated that transgene expression occurred in the principal cells of the mid/distal caput epididymidis, thereby mimicking the spatial endogenous mE-RABP gene expression. Transgene and mE-RABP gene expression was detected from 30 days and progressively increased until 60 days of age. Castration, efferent duct ligation, and hormone replacement studies demonstrated that transgene expression was specifically regulated by androgen but not by any other testicular factors. Altogether, our results demonstrate that the 5-kb promoter fragment of the mE-RABP gene contains all of the information required for the hormonal regulation and the spatial and temporal expression of the mE-RABP gene in the epididymis.

Androgens↗
PubMed1999

Synthesis of retinoic acid by rat ovarian cells that express cellular retinoic acid-binding protein-II.

The induction of pseudopregnancy by the injection of eCG in rats results in the appearance of cellular retinoic acid-binding protein type II (CRABP[II]) in the granulosa cells of the ovary and the lining epithelium of the uterus within 48 h. This expression pattern is also seen in the normal mature female rat, in which CRABP(II) is expressed in the uterine epithelium during estrus (but not diestrus) and in the granulosa and luteal cells of the ovary. We have previously demonstrated that the uterine epithelial cells from the pseudopregnant rat have gained the ability to synthesize retinoic acid from retinol, in correlation with the induced expression of CRABP(II). If this is true for other sites of CRABP(II) expression, then local production of retinoic acid is intimately connected with various stages of reproduction in the female. Here we report that granulosa cells from the ovary of the eCG-treated immature rat and luteal cells from the ovary of the eCG/hCG-treated immature rat (both of which express CRABP[II]) synthesized markedly higher amounts of retinoic acid when cultured, compared to granulosa cells cultured from the ovary of the prepubertal rat treated with control vehicle. Culturing the granulosa cells from either control or eCG-treated animals had no effect on the expression of CRABP(II) cells. These data are consistent with our hypothesis that CRABP(II) expression is associated with retinoic acid synthesis and strengthen the case that local generation of retinoic acid plays an important role in reproduction.

Animals↗
PubMed1998

Spatial and temporal patterns of expression of cellular retinol-binding protein and cellular retinoic acid-binding proteins in rat uterus during early pregnancy.

Retinoic acid, perhaps the most potent hormonal form of the naturally occurring retinoids (retinol and derivatives), is required in vivo for the maintenance of normal pregnancy and embryo development. However, little is known about the specific sites of action and metabolism in the uterus during pregnancy. In this study we describe the pattern of temporal and cell-specific expression of cellular retinol-binding protein (CRBP) and cellular retinoic acid-binding proteins type I and type II (CRABP and CRABP[II], respectively) in the rat uterus during the periimplantation period (Day 1 to Day 7 of pregnancy; Day 1 = presence of vaginal plug). Immunohistochemical studies showed that there were dramatic and rapid changes in expression pattern of the retinoid-binding proteins after mating as early as Day 1, as well as a differential expression of these proteins when the mesometrial side and antimesometrial side of the uterus were examined during the periimplantation period. CRABP(II), whose presence has been previously shown to correlate with retinoic acid synthesis in the uterine epithelium, was specifically localized to the luminal epithelium at Day 1, being stronger on the mesometrial side, and then fell to lower levels. CRBP was also expressed in the luminal epithelium on the mesometrial side at Day 1 as well as in some stromal cells, declining at these sites over the next several days. CRABP was localized to some of the stromal cells at Day 1, overlapping CRBP expression. Embryonic implantation was accompanied by the appearance of CRBP and CRABP(II) in the decidual cells. CRBP and CRABP were also present in both smooth muscle layers of the uterus. The changes in the temporal and cell-specific distribution of retinoid-binding proteins imply a multifunctional role of vitamin A in uterine cell proliferation, differentiation, and embryonic implantation. The presence of CRABP(II) suggests that local generation of retinoic acid is important in these processes.

Animals↗
PubMed1998

Molecular cloning and hormonal regulation of a murine epididymal retinoic acid-binding protein messenger ribonucleic acid.

A complementary DNA encoding the mouse epididymal secretory protein MEP 10 (mouse epididymal protein 10) was cloned and is now renamed murine epididymal retinoic acid binding protein (mE-RABP). The analysis of the predicted primary amino acid sequence showed that mE-RABP has a 75% identity with rat ESP I (epididymal secretory protein I), another epididymal retinoic acid-binding protein. The homology strongly suggests that mE-RABP is the mouse orthologue of rat ESP I. A computer analysis of the predicted three-dimensional structure confirmed that mE-RABP can accommodate retinoic acid as ligand. In the rat, ESP I messenger RNA (mRNA) is expressed in the efferent ducts and in the entire caput epididymidis. However, in the mouse, the expression of a 950-bp mE-RABP mRNA was detected only in principal cells of the mid/distal caput epididymidis, suggesting that the regulation of region-specific expression is different in rat and mouse. Northern blot analyses showed that mE-RABP gene expression is no longer detected 10 days after castration but progressively rebounds between days 15 and 60. However, mE-RABP protein could not be detected by Western blot 30 days after castration. Androgen replacement, begun 5 days after castration and continued for 4 days restored significant expression of mE-RABP mRNA. Efferent duct ligation for 10 days did not affect gene expression. Taken together, these results indicate that mE-RABP mRNA expression is regulated by androgens but not by testicular factors. The overall similarity in the primary amino acid sequence of mE-RABP with ESP I and other members of the lipocalin superfamily suggests that they are evolutionarily related.

Amino Acid Sequence↗
PubMed1997

Cellular retinoic acid-binding protein(II) presence in rat uterine epithelial cells correlates with their synthesis of retinoic acid.

Vitamin A (retinol) and retinoic acid are necessary for the maintenance of the female reproductive system of higher animals. Our previous work has demonstrated cell specific expression of cellular retinoic acid-binding protein (CRABP) and cellular retinoic-acid binding protein(II) [CRABP(II)] in the uterus of the rat. CRABP(II) expression was shown to be induced in the uterine surface epithelial cells by treatment of prepubertal rats with pregnant mare serum gonadotropin (PMSG). Here we report that, after PMSG treatment, collected uteri had markedly higher levels of retinoic acid than did the uteri of prepubertal rats treated with the control vehicle. Smooth muscle, stromal, and epithelial cells were then cultured from uteri from such animals and provided with retinol or with the retinol/retinol-binding protein complex. Retinoic acid production, analyzed by high-performance liquid chromatography, was observed for the epithelial cells from the uteri of prepubertal animals treated with PMSG, cells previously shown to express CRABP(II) and confirmed here to continue to express it in culture. Little or no retinoic acid was produced by cultured epithelial cells from the prepubertal uteri [shown previously to be negative for CRABP(II)] or by smooth muscle and stromal cells taken from uteri of prepubertal or PMSG-treated rats (shown previously to express CRABP). Retinoic acid production by uterine epithelial cells [and CRABP(II) expression] was also observed if the prepubertal rat was treated with estrogen before cell collection. At no time did cells expressing CRABP exhibit significant retinoic acid synthesis. Thus, this system revealed an important difference in retinoid metabolism between cells expressing CRABP and CRABP(II) and suggests CRABP(II) may participate in retinoic acid production and/or secretion.

Animals↗
PubMed1997

Variable expression of cellular retinol- and cellular retinoic acid-binding proteins in the rat uterus and ovary during the estrous cycle.

Ovaries and uteri from normal adult female rats at known stages of the estrous cycle were analyzed for the presence of cellular retinol-binding protein (CRBP) and both types of cellular retinoic acid-binding protein (CRABP and CRABP II). Northern and Western blot analysis of the uteri revealed a peak of CRBP during diestrus and a peak of CRABP during proestrus, whereas CRABP II peaked sharply during estrus. Immunohistochemical studies showed CRABP II localized to the luminal epithelium, while both CRBP and CRABP were observed only in the smooth muscle layers of the uterus. In the ovary, CRABP was not detected, while CRBP levels remained relatively constant throughout the cycle and CRABP II peaked slightly during metestrus. CRBP in the ovary was localized to the oocytes, nearby granulosa cells, and some regions of stroma. CRABP II was found predominantly in the granulosa cells of mature follicles and early corpora lutea, as well as some regions of the stroma. These results suggest a need for further studies to assess the role of retinol and its metabolites in normal uterine function and ovarian follicular development.

Animals↗
PubMed1996

Localization of cellular retinoic acid-binding protein (CRABP) II and CRABP in developing rat testis.

Retinoic acid (RA) has been implicated as a signaling molecule for the morphogenesis of some tissues and organs. The morphogenesis of the rat testis occurs relatively late in development, culminating in puberty. Two members of the superfamily of small intracellular carrier proteins for lipophilic compounds are cellular Ra-binding protein (CRABP) and cellular RA-binding protein II (CRABP-II). Both CRABP and CRABP-II are present at various sites in the developing mouse embryo. Here we report the developmental expression and localization of CRABP and CRABP-II in rat testis. Northern blot analysis of CRABP-II demonstrated the highest messenger RNA expression on day 4 (the earliest time point assayed by this technique), decreasing thereafter until day 20, when it became undetectable. Western blot analysis, begun on day 19 of fetal development, indicated that high levels of protein expression in the testis already existed at that time. CRABP messenger RNA expression reached its highest levels between postnatal days 16-20 and decreased thereafter. Immunolocalization revealed that CRABP-II was confined to the fetal population of Leydig and Sertoli cells. We observed that CRABP-II was expressed in certain cells that synthesized retinoic acid in the uterus and ovary (unpublished). The expression of CRABP-II in Sertoli cells and fetal Leydig cells suggested that these cells may well be the site of RA synthesis in the developing testis. CRABP was localized to gonocytes in earlier stages and spermatogonia later, where it was clearly excluded from the nucleus, indicating that the role of CRABP may be to protect these cells from the effects of RA. The reported expression of CRABP-II in embryonal tissues, which are RA responsive and undergoing morphogenesis, coupled with CRABP-II expression in the testis at a critical morphogenic stage suggest that RA may play a prominent role in the morphogenesis of the testis.

Aging↗
PubMed1996

Regulation and localization of cellular retinol-binding protein, retinol-binding protein, cellular retinoic acid-binding protein (CRABP), and CRABP II in the uterus of the pseudopregnant rat.

Three members of the superfamily of small intracellular carrier proteins for lipophilic compounds are cellular retinol-binding protein (CRBP), cellular retinoic acid-binding protein (CRABP), and cellular retinoic acid-binding protein II (CRABP II). Retinol-binding protein (RBP) is a secreted protein that binds and solubilizes vitamin A for transport. Here we report the coordinate regulation of RBP, CRBP, retinol, and CRABP II in the uterus of the pseudopregnant rat. In the proliferative stage of the uterus, which was induced by PMSG, the messenger RNA (mRNA) and protein levels of RBP and CRBP as well as retinol levels significantly decreased. This pattern of regulation was duplicated by estrogen treatment of prepubertal rats. In addition, CRBP and RBP were found to be colocalized to the stromal cells of the rat uterus by immunohistochemistry and [35S]methionine-labeled affinity chromatography, respectively, and were not detected in other cell populations. CRABP II mRNA and protein expression were up-regulated in the proliferative phase of the uterus brought about by PMSG injection or, alternatively, by estrogen treatment of prepubertal rats. CRABP II was localized to the surface epithelium, but was not seen elsewhere, including glandular epithelium. Immunolocalization of CRABP showed staining of the smooth muscle and stromal cells of the uterus. The appearance of CRABP in the stroma of the uterus also correlated with PMSG injection as well as estrogen treatment. Although estrogen induced the appearance of both binding proteins, CRABP mRNA levels peaked between 4-24 h postestrogen treatment, whereas CRABP II mRNA levels continued to rise 48 h postestrogen treatment. These data demonstrate an important role for vitamin A and retinoid-binding proteins in rat uterine physiology.

Animals↗