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Correspondence |
, and Estrogen Receptors: Possible Cross-Talks and Interactions
University of Porto Porto, Portugal
To the Editor-in-Chief:
Recently, we read with special interest the paper published by Graubert et al1 , concerning the modulation of vascular endothelial growth factor (VEGF) during the menstrual phases. The authors made several statements regarding the VEGF mRNA levels in endometrial stromal cell cultures submitted to different estrogen and progesterone in vitro treatments. They concluded that hypoxia induced a 2.4-fold increase in VEGF mRNA levels by 48 hours of exposure, estrogen and progesterone stimuli slightly raised the VEGF mRNA levels, and no decrease in VEGF mRNA was observed after withdrawal of the estrogen and progesterone.1 In addition, the authors also infer that it is unlikely that steroids play a direct role on VEGF regulation, a view that is controversial, and in contrast with our findings in breast epithelial cells.2,3
We have been studying the effects of estrogen and progesterone in the expression of VEGF mRNA and protein using a human breast cancer cell line, MCF-7 (ATCC). Briefly, after culturing MCF-7 breast cancer cell line for 48 hours with 17ß-estradiol 1 x 10-9 mol/L (Sigma) or progesterone 1 x 10-8 mol/L (Sigma), we evaluated the expression of mRNA and protein levels of angiogenic factors, namely VEGF, by RT-PCR and Western blotting, respectively. Whereas Graubert et al1 observed only a slight increase in VEGF transcript after estrogen stimulation, our preliminary results demonstrate that, somehow, estrogen induces VEGF overexpression, both in mRNA and protein levels. In accordance with our results, a recent report showed the presence of estrogen response elements in VEGF gene promotor region,4 indicating that estrogens are, in fact, involved in VEGF up-regulation.
In the last two years, several papers concerning the different
patterns of estrogen receptor (ER) 
and ß expression by
epithelial, stromal, and vascular endothelial endometrial cells have
been published.5–8
Mueller et all5
and Lecce
et al6
showed a highly complex pattern of and ß
receptor distribution during the menstrual cycle. It has been shown
that ER- and ER-ß mRNA levels in the eutopic endometrium were
affected by a cycle change in ovarian hormones.7
We would
be interested in knowing the estrogen receptor profile of those stromal
endometrial cells during the cell culture, since no basal tonus
hormonal stimulation was maintained during the experiment, or at least
it is not shown. This might lead to a down-regulation of the estrogen
receptors, since their expression is transient during the menstrual
cycle and is highly dependent on the estrogen, progesterone,
luteinizing hormone, and follicle stimulating hormone
levels.7,8
A distinct pattern of ER among breast and
endometrium tissues would also explain the discrepancy between the
Graubert et al1
results and ours. It is also known that
different ER modulators (both ER coactivators and corepressors) are
differentially expressed within different organs, which would lead to
different responses after estrogen stimulus.
Conversely, the authors showed a moderate increase in VEGF mRNA levels after progesterone treatment;1 these findings are very similar to what we observed in MCF7 in vitro experiments. VEGF is involved in proliferation and migration of vascular endothelial cells. Since progesterone is mainly synthesized during endometrial secretory phase, this steroid hormone is likely to mediate the growth and maintenance of stable coiled arterioles that characterize this phase, through the activation of growth factors other than VEGF.
Moreover, these authors did not find any increase in mRNA levels of
TGF- and IL-1ß when the endometrial cells were submitted to
hypoxic stimulus.1
We also evaluated the hypoxic effects
in MCF-7 cultures using a different model (hypoxia-like effect induced
by CoCl2 added to culture medium). Despite the
differences in our model and the one used by the authors,1
our results were very similar concerning the TGF- mRNA and protein
levels. In fact, TGF- expression was not induced by hypoxic
conditions in MCF-7 cells.
Our group has previously reported that TGF-, a growth factor
activated by estrogen,2,3
associated with higher
angiogenic rates in a series of 86 invasive breast cancer
cases.2,3
Since ER- is the predominant activated
isotype in breast tissue, and in agreement with TGF--driven VEGF
up-regulation reported by Graubert et al1
in stromal
endometrial cells, we can hypothesize that ER- activated on estrogen
stimulus might promote TGF- expression, which up-regulates VEGF.
This putative mechanism defines a relevant role of estrogen in
angiogenic switch. However, further studies are needed to reach a
conclusive model of ER-, ER-ß, TGF-, and VEGF cross-talk.
|
and angiogenesis. Am J Surg Pathol 1999, 23:358-359[Medline]
, a missed link? The Breast 1999, 8:154-156
, and Estrogen Receptors: Possible Cross-Talks and Interactions
*University of California Los Angeles, Los Angeles, California
Palmetto Fertility Center of South Florida Miami, Florida
Authors’ Reply:
We thank the group at the University of Porto (Portugal) for their interest in our work and for bringing to discussion some important aspects related to regulation of VEGF by steroids, a much-debated issue.
In our recently published manuscript1
we described
consistent, but modest, increases in VEGF mRNA levels under culture
conditions when exposed to steroids. This was contrasted by the effect
of hypoxia and other cytokines (IL-1 and TGF-), which elevated VEGF
mRNA nearly 10-fold. Results from Northern blots of total endometrial
tissue using human subjects with hormonal determination of cycle stage
revealed that overall VEGF mRNA is significantly increased upon
menstruation, a time when both estrogen and progesterone levels are
lowest and hypoxia is highest. The combination of these results and
much validation from in vitro experiments supported our
general conclusion that "it is unlikely that sex steroids play a
significant role on VEGF regulation during postmenstrual repair as
circulating estrogen and progesterone levels are physiologically low at
this point in the cycle" (Am J Pathol
158:1408). If one is to focus attention on the proliferative and
secretory phases, results from our Northern analysis support that VEGF
is increased by 1.6- and 1.8-fold, respectively, considering 1-fold
levels in early proliferative phase. Whether steroids alone are
responsible for these increases requires further investigation.
More revealing and pertinent to the discussion at hand are results from
in situ hybridization. Evaluation of VEGF transcripts in the
endometrium of women during the proliferative phase showed low levels
in the glands (Figure 1
A and B, open
arrows) and higher expression in the
stroma (closed arrows). In contrast, secretory endometrium showed
strong transcript levels in the glands (open arrows) with light
expression in the stroma (C and D, closed arrows). These results were
not included in our manuscript because the major conclusions have been
previously published by another group using immunocytochemistry, but
with identical results.2
The take-home message is that
different cellular compartments respond differently to the same
hormonal levels. Naturally it is the combination of multiple signaling
pathways and their integration that results in variations of transcript
levels. We feel that keeping this in mind is essential for
interpretation of in vitro data.
The group of Porto reports unpublished information using MCF-7 cells. We do not argue with their findings. In fact, increases of VEGF by estradiol has been reported by several groups (a brief evaluation shows 12 published papers centered on this subject alone). Our overall assessment from working on this problem and closely following the literature is that different cells will respond differently to similar signals and this appears to be the case with VEGF. In addition, in vivo validation is essential to ascertain the biological relevance of in vitro findings. In this light, in a recent publication in AJP, using the VEGF promoter linked to GFP might prove to be of extreme value.3 Not to be repetitive, we would like to direct interested readers on the subject to a well put together commentary by Drs. Sengers and Van De Water4 on that same issue.
Finally, thanks to the editorial panel of The American Journal of Pathology for providing us with the opportunity to present our response and to Dr. Larry Brown (Department of Pathology, BIDMC, Boston, MA) for allowing to use his in situ data as part of this response.
References
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