VCAM-1 and a4 integrins have been shown to play important roles in targeting immune cells to sites of injury (Dämle and Aruffo, 1991) as well as in immune cell differentiation (Miyake et al., 1991b; Ryan et al., 1991). The more recent demonstration of the importance of their interaction during skeletal myogenesis (Rosen et al., 1992), suggested that VCAM-1 and a4 integrins might play other important developmental roles during embryogenesis. We began this project in search of novel expression patterns of these two counter-receptors, and found their interaction to be vital to more than hematopoiesis and skeletal myogenesis. Here we report the spatiotemporal expression of VCAM-1 and a4 integrin in several aspects of murine development, including differentiation of the neuroepithelium of the CNS, adhesion of the placenta, and especially morphogenesis of the heart. We have also targeted the heart, or more specifically the myocardium, to examine the expression of VCAM-1 during a morphogenetic event, and to examine the elements responsible for the pattern of expression so important to cardiogenesis.
We have examined the patterns of VCAM-1 and a4 integrin expression in embryonic and adult mouse tissues. As expected, VCAM-1 and a4 were found at embryonic sites of hematopoiesis such as liver and yolk sac, just as they are expressed in the adult bone marrow: a4 was found on maturing immune cells and on undifferentiated cells, whereas VCAM-1 was present on stromal cells. Suggesting that these receptors could have parallel roles in embryonic and adult hematopoiesis. Also corroborating previous work, VCAM-1 and a4 were identified on all differentiating skeletal muscle, supporting their role in skeletal muscle differentiation.
We found that a4 is also expressed on vascular smooth muscle. However, unlike skeletal muscle, a4 was present on vessels very early during embryogenesis and it persisted into adulthood. Similarly, a4 was found on smooth muscle throughout the digestive tract. Also, in contrast to skeletal muscle, VCAM-1 was not present on any normal smooth muscle examined, except, notably, for that of the stomach. Although VCAM-1 was not evident on vessels in the lung, it was present along with a4 on surrounding mesenchyme, which gives rise to the vascular endothelium and smooth muscle. It is still unknown whether an interaction between these receptors is in any way involved in pulmonary morphogenesis.
Unexpected were the findings of VCAM-1 and a4 on the non-hematopoietic extraembryonic membranes, CNS and heart. The expression of VCAM-1 on the developing allantois with the adjacent expression of a4 on the chorionic plate was made particularly interesting by the lack of placentation in many of the embryos lacking either receptor (Gurtner et al., 1995; Kwee et al., 1995; Yang et al., 1995). On the other hand, the expression of VCAM-1 on the neuroepithelium and radial glia of the developing CNS is still something of a mystery, since none of the VCAM-1 deficient embryos survived to an age at which effects on the CNS could be detected. However, the work done in our lab on differentiating P19 cells following this discovery has been vital to much of what has been determined about VCAM-1 expression in the heart.
On the E7.5 heart, our earliest time point, immunolabeling for VCAM-1 was visible throughout the forming myocardium. This expression coincides with the expression of myosin and desmin in the myocardium previously described in rat embryogenesis (Baldwin, et al., 1991), suggesting that the VCAM-1 gene is activated quite early during the differentiation of cardiomyocytes. Later, around E9, a4 expression appears on the dorsal mesocardium as well as on extending villi from which mesocardial cells migrate across the outer surface of the compact myocardium to form the epicardium. Concurrently, VCAM-1 expression was seen to increase on the compact myocardium relative to the trabeculae. The most obvious conclusion, that these receptors play a role in formation of the epicardium, is supported by VCAM-1 and a4 knock-out studies, in which mice lacking either gene formed incomplete epicardia (Baldwin et al., 1994b; Kwee, et al., 1995; Yang, et al., 1995).
Between E10 and E16, localization of VCAM-1 expression becomes increasingly restricted to the muscular ventricular septum. Again, the importance of VCAM-1 in the genesis of the muscular ventricular septum is confirmed by VCAM-1 knock-out studies, in which mice lacking the VCAM-1 gene die by E12 with underdeveloped, or in some cases no, ventricular septa (Kwee et al., 1995). The observations that VCAM-1 functions in the septum in the absence of a4 and that VCAM-1 is expressed differentially on cardiomyocytes suggest an uncharacterized role for VCAM-1 in the heart. This made the myocardium particularly interesting as a novel tissue in which to study the activities of the VCAM-1 promoter in order to determine how this differential expression pattern is maintained.
To examine the elements responsible for the expression of VCAM-1 on the developing myocardium, we first had to convert from mounted embryos to tissue culture, so that we could more easily manipulate the cardiomyocytes. We verified that the differential expression of VCAM-1 could be maintained in culture on primary cardiocytes from E18 embryonic rat hearts. More importantly, we found that the H9c2(2-1) cell line was sufficiently similar in its expression of VCAM-1 to serve as a model for embryonic myocardium. We were then able to subclone the H9c2(2-1) cells, and isolate colonies that were either positive or negative for VCAM-1 expression, giving us an opportunity not available with the primary cardiomyocytes to examine this difference in expression.
With culturable cells, it became possible to examine the activities of VCAM-1 promoter / CAT reporter constructs in the context of cardiomyocytes. Primary rat cardiomyocytes, H9c2(2-1) cells, and each of the subcloned lines were transfected with the VCAM-1 promoter constructs. The data from these experiments demonstrated that while the 32 bp core promoter yielded basal level expression, a region between -2.1 kb and -288 bp upstream of the VCAM-1 promoter was acting as a silencer in the mixed populations, as well as in the VCAM-1 negative cells. Conversely, the -2.1 kb fragment and -288 bp fragment was almost equally active in the VCAM-1 positive subclones, similar to the activities of this construct in the C2C12 skeletal myoblast cell line (Jesse et al., 1998). By further truncating the upstream fragments of the VCAM-1 promoter and placing these fragments in front of an SV40 promoter, we were able to map the silencer activity of this region to an octamer site 1180 upstream of the transcription start site.
Octamer sites had been previously identified as silencers in the region between -288 bp and -2.1 kb in the context of neural differentiation of P19 cells specifically at 1180 and 1554 bp upstream of the transcription start site (Sheppard, et al., 1995). Octamer sites are the binding sites for POU-domain homeotic transcription factors, which can sometimes act as repressors of transcription (Jacobson, et al., 1997). Also within this region are elements which weakly match Retinoic Acid Response Elements (RARE's). Although expression of the retinoic acid receptor, RARa, in the heart precedes the downregulation of VCAM-1 on the myocardium by several days, it is worth noting, that experiments knocking out RARa result in a malformation of the compact myocardium and ventricular septum (Kastner, et al., 1994) quite reminiscent of the VCAM -/- phenotype. In fact, the work that identified the octamer elements as silencers in the VCAM-1 promoter also showed induction of VCAM-1 expression on P19 embryonal carcinoma cells several days after retinoic acid treatment (Sheppard, et al., 1995), suggesting that a possible link between retinoic acid induction and VCAM-1 expression may be part of a common differentiation mechanism. While attempts to date at linking any of these potential RARE's with silencing or activation of the VCAM-1 promoter suggest that they are non-functional, other studies have indicated a direct correlation between the activities of various POU proteins and retinoic acid induction (Ryan et al., 1997).
Since the -1180 octamer site appeared to contain the activity responsible for the differential expression of VCAM-1 on cardiomyocytes, it was on this element that further studies were focused. When used as a probe for gel shift assays, this element demonstrated higher affinity for bound protein complexes than any of the other potential octamer elements, and the same major complex could be formed in extracts made from hearts dissected from rat embryos at E11 and E16, from primary cardiomyocytes, as well as in H9c2(2-1) cell extracts. While this suggested that the -1180 octamer element was binding relevant proteins, it wasn't until we began to dissect these complexes that the real story began to unfold.
We used antibodies against several known POU proteins, and found that antibodies against Oct-1 could supershift the major band in each of the extracts tested. Unfortunately, the ubiquitous nature of Oct-1 expression suggested that an auxiliary protein, like Bob-1, might be the actual controller of VCAM-1 expression: however, our Bob-1 antibody was unable to shift or block any of the complexes visible on our assays - probably because of inaccessibility of the epitope in the particular complexes present in heart tissue.
Not to be deterred, we performed western blot analyses of each extract using antibodies against our entire panel of POU proteins and Bob-1. The only two of these proteins we found to be present were Oct-1 and Bob-1, which were present in each tissue as well as in the H9c2(2-1) cell line. Furthermore, immunostaining of mouse hearts from E8 through E18 showed Bob-1 expressed evenly throughout the myocardium, even as VCAM-1 became increasingly restricted to the interventricular septum. So Bob-1 expression correlated with the expression of VCAM-1 during early cardiogenesis, although it is interesting to note that Bob-1 expression remained constant throughout cardiogenesis, even as VCAM-1 expression became progressively restricted, suggesting that while Bob-1 may well be responsible for the upregulation or enhancement of VCAM-1 expression in the forming myocardium, it is the influence of a repressive transcription factor separate from Bob-1 (but binding the same octamer element) that effects its progressive downregulation.
This still didn't demonstrate whether Bob-1 could bind to the VCAM-1 promoter, so we biotinylated our -1180 probe and used it to precipitate proteins extracted from embryonic murine heart tissue. Two proteins so precipitated were identified by Western blot as Oct-1 and Bob-1. Now finally, we had in Bob-1 a transcription factor that bound to the -1180 octamer element and appeared to colocalize with VCAM-1 expression throughout cardiogenesis. But, we still needed to confirm the ability of Bob-1 to effect the transcription of VCAM-1.
With the availability of Bob-1 expression constructs, we were able to overexpress Bob-1 in H9c2(2-1) cardiomyocytes and found that this overexpression could overcome the silencing of the VCAM-1 promoter by the upstream octamer sites, as compared to control constructs. More importantly, extracts from Bob-1 overexpressing cells demonstrated an increase in endogenous VCAM-1 protein expression. When we went back to our constructs placing the octamer sites upstream of the SV40 promoter, we found that the overexpression of Bob-1 could not only derepress, but could also activate constructs containing octamer sites above controls.
The ability of Bob-1 to activate VCAM-1 expression in cardiomyocytes and its overlapping expression on the myocardium during development, point to Bob-1 as a major factor in the upregulation of VCAM-1 during the early stages of cardiogenesis. The fact that Bob-1 remains high throughout the myocardium while VCAM-1 becomes increasingly restricted to the interventricular septum suggests that while Bob-1 is responsible for the upregulation of VCAM-1 throughout the myocardium, there must be another, as yet undefined, transcriptional repressor that binds the same octamer sites as the Bob-1/Oct-1 complex. This repressor appears to both deactivate Bob-1 and repress the basal promoter. If the previous studies are true, and the RARE's are indeed non-functional, then this would suggest that the repressor be another POU protein.
Future Directions
It is worth noting that Bob-1 expression has not been previously described in the heart. Although it is possible that Bob-1 expression in the myocardium exists solely to promote VCAM-1 expression, it is more than likely that other proteins involved in heart formation are influenced by Bob-1 expression. Mice lacking the gene for Bob-1 have been generated, and these mice appeared to be healthy and phenotypically unchanged outside of reduced germinal centers in the lymph nodes (Schubart et al., 1996; Kim et al., 1996). While VCAM-1 is expressed in the lymph nodes (see above), it is doubtful that VCAM-1 plays any part in this obviously lymphoid degeneration. It is more likely the lack of mature B-cells that is responsible for this phenotype, especially since each group working with the Bob-1 deficient mice also reported diminished B-cell activity (Schubart et al., 1996; Kim et al., 1996). It has been suggested, regarding this lack of gross phenotypic changes in the Bob-1 deficient mice, that there may be a redundant factor equivalent to Bob-1 which can compensate for its loss (Kim et al., 1996). It is also possible that removing Bob-1 may alter expression of Oct-1 such that this factor can compensate for its diminished ability to activate promoters.
A better way to test the importance of Bob-1 in development might be to overexpress it using a heart-specific promoter (Ross et al., 1996). Our data predict that overexpression of Bob-1 in the developing myocardium could result in an overabundance of cardiomyocytes within the walls of the heart, resulting in hypertrophy. This prediction comes from the observations made in cardiomyocytes as well as neuroepithelial cells, that VCAM-1 expression coincides with proliferation. More specifically, it appears that in either the heart or the CNS, those cells expressing VCAM-1 represent a proliferative population, while the loss of this expression of VCAM-1 appears to follow differentiation. Although no direct link between VCAM-1 expression and proliferation has yet been established, it is intriguing to think that VCAM-1 expression is restricted to the ventricular septum because it is the most actively growing portion of the myocardium during late development. If the theory is correct, the overexpression of Bob-1 on the myocardium should result in the derestriction of VCAM-1 expression across the myocardium, which should result in continuous proliferation of cardiomyocytes and eventual hypertrophy.
This suggests another potential project that might elucidate any connection between VCAM-1 expression and proliferation. For these experiments, a VCAM-1 expression vector similar to the Bob-1 expression vectors would be used to transfect H9c2(2-1) cells, or possibly primary cardiomyocytes. After transfection, the growth rate of transfected and untransfected cultures would be monitored by direct observation of cultures and counting trypan blue stained, suspended cells. The advantage of using a VCAM-1 expression vector in cultured cells, is that if promising results are attained and it looks like a correlation might exist, further manipulations of the transfected cell, like maintaining the cultures in suspension or low-serum media or even soft agar, might extend our findings to identify the conditions under which VCAM-1 expression might induce proliferation.
A more difficult, and potentially less fruitful project would be to scale up the octamer binding assays, in an attempt to identify the repressor protein(s) responsible for the restriction of VCAM-1 during ventricular septation. A more powerful method for identifying this factor may be to use the octamer element in a "One-Hybrid" assay. In this assay, the relevant promoter fragment is placed upstream of the reporter, and cotransfected with a cDNA library attached to an activator (usually VP16). If cDNA prepared from embryonic heart were used for this assay, the resultant positive clones should represent transcription factors able to bind the target site. These clones could then be used in complementation studies along with the Bob-1 expression vectors to identify proteins able to deactivate the VCAM-1 promoter in the presence of Bob-1 - which would correlate to the balance between these transcription factors during the latter parts of murine cardiogenesis.
The discovery of Bob-1 expression on the myocardium and the correlation of this expression with the expression of VCAM-1 on cardiomyocytes suggest that Bob-1 may play a role in the expression of other factors that are important for normal heart formation. Certainly, this open the door to further research into the possible functions of Bob-1 in the heart, as well as suggesting a novel mechanism for genetic activation in developing cardiomyocytes.
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