Transcriptomic analysis of two major signaling pathways of the TGF-β superfamily unexpectedly showed few, poorly annotated Smad4-independent genes (Fig. and ). Previously, fibroblasts from Smad4
knockout mice gave evidence for a more significant Smad4-independent cell response (37
). However, the physiological effects of TGF-β superfamily members differ dramatically between mesenchymal and epithelial cells, making it difficult to compare studies on such disparate cell types. Our findings agree with independent epithelial cell studies using mutant TGF-β receptors unable to activate the Smad pathway (13
) and are in favor of the central role of Smad4 in all TGF-β superfamily pathways (33
). The common genomic program regulated by TGF-β1 and BMP-7 is rather short and includes genes implicated in cell proliferation control (p21Cip1
; Fig. ). In addition, we identified several novel gene targets of both pathways in epithelial cells, such as ovol1
, and hsd17b2
. These genes may be of importance for understanding distinct functions of the two signaling pathways and attract our future focus. Our microarray screen revealed 129 genes regulated specifically by one of the two growth factors (Fig. ). It is possible that among them are critical genes that define the final and specific effect of TGF-β1 or BMP-7 on epithelial cells. However, as we have shown for Id2
, differential regulation of common gene targets by TGF-β and BMP can explain distinct physiological effects of these two pathways.
We focused on Id2
because of their robust gene regulation (Fig. ), and due to the limited understanding of their role in TGF-β physiology, while Id1 is the best-analyzed Id member in terms of its regulation by BMP and TGF-β, it remains unclear how it contributes to cell proliferation control by these factors (14
). Induction of Id1
by BMP-2 in breast cancer cells or by TGF-β1 in fibroblasts was recently shown (3
). Studies of lymphocyte differentiation provide convincing evidence for Id2 and Id3 roles in TGF-β physiology (12
). Using a panel of normal epithelial cells we establish here a general profile of Id2
regulation (Fig. and ; also see Fig. S1 and S2A in the supplemental material). This includes the MDA-MB-468 mammary carcinoma cell where the initial microarray screen was performed, whereby Id2
scored as positively regulated genes at the early time point of TGF-β stimulation (Fig. ). Detailed immunoblot analysis and time course experiments showed that this cell line, like more normal epithelial cells, also responds to TGF-β after Smad4 reconstitution, by eventually downregulating Id2 and Id3 levels at late time points. On the other hand, we still do not understand why TGF-β can transiently induce Id2
mRNA but not protein expression in certain cell types (see Fig. S1 in the supplemental material) and both Id2/Id3 mRNA and protein levels in MDA-MB-468 carcinoma cells (Fig. ). Our focus on the functional role of Id2 and Id3 regulation downstream of TGF-β and BMP signaling led to the finding that these genes define specificity of the two pathways in epithelial cell biology.
Since knockdown of Id2 (Fig. ) and Id3 (Fig. ) enhances growth inhibition by TGF-β1 and BMP-7, we consider these proteins to be important components controlling cell proliferation by TGF-β members. It would be interesting to test whether loss of Id1 function could have similar effects on TGF-β/BMP-mediated epithelial growth inhibition. Throughout this study we observed that Id2 was always more efficient to regulate cell growth in response to TGF-β members, when compared to Id3 (Fig. and ). This cannot be due to the expression levels of the two proteins as the same result was reached when high ectopic levels of each Id member were expressed in various epithelial cell models. Finally, when both Id2 and Id3 were ectopically expressed, we never observed additive or synergistic effects compared to experimental perturbation of each Id member separately (Fig. ). These observations suggest that Id2 and Id3 have common targets in epithelial cells, e.g., common bHLH proteins which regulate the same critical target genes (see below). If each Id protein had distinct targets that were cooperatively contributing to the control of cell proliferation, then we should have observed at least additive if not synergistic effects. It is therefore possible that the higher efficiency by which Id2 blocks TGF-β-mediated physiological effects reflects a posttranslational modification which is directly induced by the TGF-β signal. Such modification could either enhance the inhibitory activity of Id2 or inversely dampen the activity of Id3. Alternatively, a more likely scenario is the possibility that Id2 modulates E-protein transcriptional activity more efficiently compared to Id3, either because of higher affinity for partner bHLH members or because of more efficient recruitment of cofactors that help mediate a complete repression of E-protein activity. Both alternative scenarios are worth investigating in the future.
Two prominent models explain the effects of Id members on cell cycle regulation (29
). Id proteins inhibit the transcriptional activity of E-proteins such as the products of the E2A
gene, E12 and E47, which positively regulate the cell cycle regulatory genes p15Ink4b
, and p21Cip1
. Alternatively, Id2 can reverse the cell cycle-inhibitory action of pocket proteins of the retinoblastoma family. Id3 cannot interact with pocket proteins, and thus, the retinoblastoma model cannot be applied to this protein. Based on our data, we suggest that TGF-β, which leads to sustained downregulation of Id2 and Id3 in epithelial cells, can block the cell cycle by the synergistic effects resulting from relief of E-protein activity on as yet unidentified gene targets and from direct transcriptional induction of the cell cycle inhibitor genes p15Ink4b
. It is worth noting here that both of these genes are induced by the TGF-β/Smad pathway directly (34
). On the other hand, BMP induces sustained Id2
expression and thus provides constant repressor function against E- and pocket proteins. BMP also induces p21Cip1
despite its positive effect on Id
expression, and downregulates c-myc
, both events favoring a halt of the cell cycle. Thus, induction of antagonistic factors by BMP may lead to almost neutral functional outcomes of cell cycle regulation in epithelial cells, which can explain why BMPs have weak and variable effects on epithelial cell growth. Considering the combined effects of TGF-β and BMP signaling on Id protein expression, we favor the model whereby Id2/Id3 repress bHLH factor activity on as yet unrecognized gene targets, distinct from cell cycle inhibitors such as p21Cip1
, whose products must be directly linked to cell cycle control. We are using cDNA microarray screens to address this important question.
The established roles of Ids as differentiation inhibitors led us to investigate such a role in response to TGF-β and BMP (Fig. and ). We focused on a mammary and a lens EMT cell model because EMT plays important roles in tumor progression (11
), and this is an epithelial response to TGF-β but not to BMP (28
). Our evidence supports a model whereby Id2/3 regulation renders TGF-β capable of eliciting EMT, whereas it waives such an instructive role from BMP. For the first time, we demonstrate that BMP induces EMT under conditions of Id2
knockout (Fig. and ). In agreement with this model BMP-7 could neutralize the EMT response to TGF-β1, since BMP-7 could override the downregulation of Id2/3 by TGF-β1 (Fig. ). Thus, during tumor progression, misregulation of epithelial Id2/3
expression could possibly allow even BMP pathways to cause EMT and enhance tumor invasiveness.
Similar to the epithelial cell growth results, the EMT data of both ectopic expression and endogenous knockdown (Fig. and ) revealed that Id2 had higher potential in blocking TGF-β-induced EMT. However, in contrast to their effects on cell growth inhibition, combinations of Id2 and Id3 ectopic expression (Fig. ) or, alternatively, combinations of Id2 and Id3 knockdown (Fig. ) demonstrated that these two proteins acted in a manner which was more than additive and possibly synergistic. This strongly implies that there exist distinct targets of Id2 and Id3 which act in a combined manner to regulate the process of EMT. We currently do not know which are the targets of Id proteins that mediate EMT. One possibility is that E-proteins such as E12 and E47, in addition to their roles in cell cycle control, may also link Id-mediated repression to EMT. This hypothesis is compatible with the known role of E2A
gene products on regulation of the E
-cadherin gene, a hallmark of epithelial differentiation (27
). However, as E-cadherin does not play a causal role in the process of EMT, there must be additional genes involved in such a process whose regulation is mediated by bHLH proteins and thus link to the action of Id2 and Id3 as described for the first time here.
We demonstrate that Id2 and Id3 are important for the concerted regulation of cell proliferation and transdifferentiation downstream of TGF-β pathways. We conclude that in epithelial tissues where multiple TGF-β ligands are active, the physiological outcome of responding epithelial cells will depend on regulation of sensitive gene targets such as Id2 and Id3. Id genes provide the first molecular targets of TGF-β pathways that explain biological specificity with respect to regulation of cell growth and differentiation.