We first examined the effects of TGFβ superfamily members on Smad activation in human keratinocytes. HaCaT cells expressed a basal level of phosphorylated Smad2 and Smad3 even in the absence of exogenous TGFβ (, left
). As expected, TGFβ treatment resulted in a robust activation of Smad2 and Smad3. In addition, and quite unexpectedly, TGFβ treatment also induced phosphorylation of the classic BMP pathway Smads, Smad1 and Smad5 (, left
). The identity of pSmad1 and pSmad5 was confirmed using a pSmad1/5-directed antibody that does not cross-react with pSmad3. Furthermore, treatment of cells with BMP2 induced phosphorylation of Smad1 and Smad5, coincident with those induced by TGFβ treatment. Conversely, activin A only activated Smad2 and Smad3 but failed to induce pSmad1 and pSmad5. The kinase inhibitor SD-093 inhibits not only the TβRI (Alk-5) kinase but also the related activin type I receptor kinases Alk-4 and Alk-7. Pretreatment of cells with SD-093 inhibited both TGFβ- and activin-induced phosphorylation of Smad2 and Smad3 (, left
). Importantly, SD-093 treatment also inhibited TGFβ-mediated induction of pSmad1 and pSmad5, indicating that their phosphorylation was dependent on TβRI (Alk-5) activity. BMP2-induced Smad1 and Smad5 phosphorylation was not affected, consistent with the fact that SD-093 does not target BMP type I receptors (8
Figure 1 Effects of TGFβ superfamily ligands on Smad activation. A, TGFβ (100 pmol/L) treatment of HaCaT cells for 1 h resulted in induction of pSmad2 (pS2) and pSmad3 (pS3) as well as of pSmad1 (pS1) and pSmad5 (pS5), all of which were blocked (more ...)
We previously described two human squamous carcinoma cell lines (SqCC/Y1 and A253) that are homozygous for missense TGFBR2
gene mutations (5
). Although endogenous autophosphorylated wild-type TβRII and transphosphorylated TβRI could be detected in HaCaT cells (5
), neither receptor species was phosphorylated in SqCC/Y1 TGFBR2E526Q
-mutant cells. On the other hand, in A253 TGFBR2R537P
-mutant cells, the levels of both TβRII and TβRI phosphoproteins were significantly higher than in HaCaT cells independently of the presence of exogenous TGFβ (5
). Thus, the TGFBR2R537P
mutation seems to constitutively activate the TβRII kinase.
To further characterize the two mutant receptors, we examined their effects on Smad activation. In SqCC/Y1 TGFBR2E526Q-mutant cells, TGFβ treatment failed to induce phosphorylation of any of the receptor-associated Smad (R-Smad) proteins (, right). In contrast, treatment with activin induced pSmad2 and pSmad3, which was inhibitable by SD-093. Similarly, treatment with BMP2 resulted in activation of pSmad1 and pSmad5, which was not affected by SD-093 treatment. Thus, in SqCC/Y1 cells, TGFβ fails to activate any of the R-Smads, whereas activin and BMP2 retained the ability to induce phosphorylation of their respective target Smads.
As in SqCC/Y1 cells, treatment of A253 TGFBR2R537P-mutant cells with TGFβ failed to induce phosphorylation of pSmad2 and pSmad3 (, middle). In contrast, A253 cells constitutively expressed high levels of pSmad1 and pSmad5 (, middle). BMP2 treatment resulted in further increasing the level of these same two phosphoproteins, which was not affected by SD-093 treatment. On the other hand, activin treatment resulted in a modest induction of pSmad2 and pSmad3, inhibitable by SD-093, but not of pSmad1 or pSmad5. Thus, neither the activin nor the BMP pathways seem to be affected by the presence of the TGFBR2R537P mutant.
To further substantiate that the bands detected by both the anti-pSmad3 and anti-pSmad1/5 antibodies in HaCaT and A253 cell extracts did, in fact, represent pSmad1 and pSmad5 and not pSmad3, we silenced endogenous Smad expression using specific siRNAs (). Silencing endogenous Smad1 and/or Smad5 expression using specific siRNAs abrogated the ability of TGFβ to induce pSmad1 and pSmad5 in HaCaT cells and resulted in the loss of the constitutively phosphorylated BMP-Smads in A253 cells (). In contrast, silencing of Smad3 did not affect the ability of TGFβ to induce phosphorylation of Smad1 and Smad5 in HaCaT cells nor did it affect the levels of phosphorylated Smad1 or Smad5 in A253 cells (data not shown). These results further substantiate that the constitutively expressed pSmads in A253 cells indeed represent pSmad1 and pSmad5 and not pSmad3.
Because SD-093 treatment did not affect the level of the constitutively active BMP-Smads in A253 cells, we investigated the possibility that the elevated levels of activated BMP-Smads might be due to a reduced rate of dephosphorylation (). As expected, TGFβ treatment induced a robust activation of Smad2, Smad3, Smad1, and Smad5 in HaCaT cells (time “0,” ). On TβRI (Alk-5) blockade using SD-093, all activated R-Smads were rapidly dephosphorylated with a half-life (t1/2
) of ~45 min, indicating that their phosphorylation state was also dependent on TβRI (Alk-5) kinase activity (). In contrast, in A253 cells, the constitutively activated BMP-Smads (Smad1 and Smad5) were resistant to dephosphorylation in response to SD-093 treatment. Similar results were obtained using two other TβRI (Alk-5/4/7) kinase inhibitors from different chemical classes, SB-431542 and NPC-30345, indicating that resistance to dephosphorylation was not unique to this particular inhibitor (; refs. 8
). Thus, constitutive activation of pSmad1 and pSmad5 in A253 cells seems to have become independent of exogenous TGFβ as well as (TβRI) Alk-5 kinase activity.
Figure 2 Dephosphorylation of activated Smads. A, assays were performed as described in Materials and Methods. In HaCaT cells, inhibition of TβRI (Alk-5) kinase activity using the selective inhibitor SD-093 resulted in dephosphorylation of activated pSmad2 (more ...)
Following 1 h of TGFβ treatment, HaCaT cell nuclei were strongly enriched for pSmad2 and pSmad3 as well as pSmad1 and pSmad5. These activated Smads underwent subsequent dephosphorylation in response to SD-093 treatment associated with a rapid reduction of total Smad2 and Smad3 levels in the nucleus, consistent with export of dephosphorylated Smads back into the cytoplasm (). Although little or no pSmad2 or pSmad3 was detected in the nuclei of A253 cells, the nuclear fraction was highly enriched for pSmad1 and pSmad5 (). In addition, SD-093 treatment had minimal effect on dephosphorylation of these activated BMP-Smads (). To determine whether the TGFBR2R537P
mutant might be responsible for the constitutive activation of BMP-Smads in A253 cells, we made use of LY2109761, a dual inhibitor that targets both the TβRII and TβRI kinases but does not affect BMP-induced cellular responses (13
). In HaCaT cells, treatment with LY2109761 caused rapid dephosphorylation of all TGFβ-activated R-Smads (). In sharp contrast to the lack of effect of SD-093 on pSmad1 and pSmad5 levels in A253 cells (), treatment with LY2109761 resulted in rapid dephosphorylation of these BMP-Smads (). Therefore, we tentatively concluded that the effects of LY2109761 on pSmad1 and pSmad5 levels were mediated by its inhibition of the TβRII receptor rather than TβRI (Alk-5), thereby resulting in dephosphorylation of pSmad1 and pSmad5.
TGFβ-induced EMT, which is an important event during wound healing, can be retained in cancer progression (14
). F-actin stress fiber formation and loss or redistribution of E-cadherin are hallmarks of EMT (14
). As expected, treatment of HaCaT cells with TGFβ resulted in actin stress fiber formation and a redistribution of E-cadherin from the cell membrane to the cytoplasmic compartment (). Furthermore, these TGFβ-induced responses were dependent on TβRI (Alk-5) kinase activity, as the phenotype could be reversed by either SD-093 or LY2109761. In fact, treatment with either of these kinase inhibitors alone resulted in a more cohesive epithelioid morphology with a more pronounced submembranous localization of F-actin and cell surface expression of E-cadherin compared with vehicle (DMSO)-treated controls. In SqCC/Y1 cells, F-actin and E-cadherin were both present at the membrane and the cells had a fairly typical epithelioid morphology (). In addition, there was no demonstrable change in phenotype in response to either TGFβ or kinase inhibitor treatment (). In contrast, in A253 cells, F-actin was diffusely expressed throughout the cytoplasm () and E-cadherin predominantly in a cytoplasmic and perinuclear pattern independently of exogenous TGFβ (). More importantly, LY2109761 treatment resulted in redistribution of F-actin toward the cell periphery and of E-cadherin to the cell membrane, whereas SD-093 had no effect. Moreover, treatment of A253 cells with LY2109761 alone (in the absence of exogenous TGFβ) induced a more cohesive epithelioid morphology with distinct cell boundaries. Thus, the TGFBR2R537P
mutant seems to endow A253 with an EMT-like state that is selectively reversible by the dual TβR kinase inhibitor and seems to be dependent on constitutive activation of pSmad1 and pSmad5.
Figure 3 Effects of TβR-kinase inhibitors on cellular responses. A, treatment ofHaCaT cells with TGFβ (100 pmol/L) for 72 h induced EMT associated with subcellular redistribution of F-actin from a submembranous location to incorporation into stress (more ...)
As shown in , TGFβ treatment of HaCaT cells resulted in a 10-fold increase in the rate of cell migration, which could be inhibited by either SD-093 or LY2109761 (). In contrast, SqCC/Y1 cells displayed very little cell migration, which was not affected by TGFβ nor by SD-093 or LY2109761 treatment (). In contrast, A253 cells displayed a very high basal rate of cell migration independently of exogenous TGFβ (). Moreover, whereas SD-093 had no effect on migration of A253 cells, LY2109761 inhibited migration by f50% (P < 0.001). With respect to invasion into Matrigel matrix, SqCC/Y1 cells were completely noninvasive for periods up to 72 h (data not shown). In contrast, A253 cells were highly invasive (). Moreover, A253 invasiveness was reduced by ~50% by LY2109761 treatment but not by SD-093 (P < 0.01; ). Therefore, it seems that TβRIIR537P- mutant cells have acquired novel properties, including constitutive EMT and high rates of migration and invasion that may contribute to the tumorigenicity of A253 in vivo. This phenotype is clearly distinct from that of the SqCC/Y1 TGFBR2E526Q-mutant cells, which seem to have lost all responses to TGFβ. Moreover, the unique in vitro properties of A253 cells were also retained in vivo. As shown in , A253 cells gave rise to poorly differentiated invasive squamous cell carcinomas in vivo. Consistent with the in vitro studies, the levels of pSmad2 expressed by the tumor cells were almost undetectable. On the other hand, the same tumor cells showed strong nuclear staining for pSmad1 and/or pSmad5, on a par with that seen in endothelial cells of the tumor-associated capillaries as well as basal and suprabasal keratinocytes in the epidermis. Interestingly, basal and suprabasal keratinocytes in the epidermis displayed the strongest pSmad1 and/or pSmad5 immunostaining, whereas cells in the stratum granulare and stratum corneum expressed pSmad2, suggesting that the two arms of TGFβ signaling might be activated at different stages of keratinocyte differentiation in vivo.
Our results suggest that TGFβ-induced BMP-Smad activation in keratinocytes involves partnering of the TβRII receptor with and activation of one of the BMP pathway type I receptors. As shown in , none of the keratinocyte cell lines (HaCaT, A253, and SqCC/ Y1) expressed Alk-1 mRNA, whereas Alk-2, Alk-3, Alk-5, and Alk-6 mRNAs were all expressed in each of the Three cell lines (). Although Alk-3 and Alk-6 receptors can activate BMP-Smads, they have not been tied to TGFβ signaling. On the other hand, as Alk-2 has been previously shown to act as a receptor for TGFβ,itis the leading candidate type I receptor to mediate the activation of BMP-Smads in HaCaT and A253 cells.
Figure 4 Detection of Alk mRNA expression by RT-PCR. Levels of mRNA expression for each of the Alk receptors were determined by RT-PCR as described in Materials and Methods. Alk-2, Alk-3, Alk-5, and Alk-6 genes were expressed in each of the cell lines to a similar (more ...)
Finally, we examined the effects of TGFBR2R537P
on coexpressed wild-type TβRII signaling. First, we compared the phenotype of FaDu × A253 somatic cell hybrid cells derived from the esophageal carcinoma line FaDu and A253 (15
) with that of the two parental cell lines. FaDu cells are null for the MADH4
gene but express wild-type TGFBR2
), whereas A253 is homozygous for the TGFBR2R537P
mutation. As shown in , treatment of FaDu cells with TGFβ resulted in activation of Smad2 and Smad3, which was inhibitable by SD-093, whereas A253 cells constitutively expressed pSmad1 and pSmad5 but failed to activate Smad2 or Smad3 in response to TGFβ. In contrast, treatment of FaDu × A253 hybrid cells with TGFβ resulted in activation of both Smad2 and Smad3 (). Most importantly, SD-093 treatment inhibited this induction, suggesting that the presence of wild-type TβRII restored TGFβ-mediated R-Smad phosphorylation in a TβRI (Alk-5)-dependent mechanism. Moreover, the levels of pSmad1 and pSmad5 seemed to be somewhat lower in FaDu × A253 than in parental A253 cells (). As shown previously, × the high levels of activated pSmad1 and pSmad5 in A253 cells were not affected by SD-093 treatment (). In contrast, in the FaDu × A253 hybrid cells, TGFβ-activated Smad2 and Smad3 underwent rapid dephosphorylation on SD-093 treatment, with kinetics similar to those observed in HaCaT (). In aggregate, these results indicate that the TGFBR2R537P
mutant is recessive over the wild-type receptor and that the associated activated cell phenotype only becomes manifest in cells that are either homozygous for the mutant receptor or have evidence of TGFBR2
loss of heterozygosity.
Figure 5 Effects of coexpression of wild-type and mutant TβRII gene. A, activation and dephosphorylation of Smads in FaDu × A253 somatic hybrid cells. Treatment of FaDu × A253 with TGFβ (100 pmol/L) for 1 h induced phosphorylation (more ...)
To further characterize the function of the TGFBR2
mutants in the context of wild-type receptor expression, wild-type and mutant TβRII were coexpressed in the TGFBR2
-null mammary cancer line T47D (10
). Cotransfection of wild-type TGFBR2
induced 3TP-lux reporter activity in direct proportion to the amount of wild-type TGFBR2
cDNA (, top left
). Thus, the TGFBR2E526Q
mutant confers a null phenotype and has no dominant-negative properties. Similarly, expression of the TGFBR2R537P
mutant by itself failed to induce 3TP-lux reporter activity. However, in the presence of both wild-type and TGFBR2R537P
-mutant receptor, the net reporter gene activity was approximately equal to that obtained with 100% wild-type receptor. Moreover, treatment with either of the two inhibitors, SD-093 or LY2109761, resulted in inhibiting 3TP-lux activity (data not shown). Essentially similar results were obtained with the SBE4-luc reporter gene construct (, top right
). On the other hand, when we used the Col7A1-luc reporter, the combination of wild-type and TGFBR2R537P-
mutant receptor seemed to have an intermediate activity, suggesting that the mutant was not contributing to Col7A1-luc induction (, bottom left
). When we examined the effects of the two TGFBR2
mutants on the TGFβ-repressed Del-5-myc
-luc reporter gene construct, coexpression of wild-type TβRII and either of the two mutants resulted in a dose-dependent decrease in Del-5-myc
-luc activity, which was proportional to the amount of wild-type receptor transfected (, bottom right
). However, in the sole presence of the TGFBR2R537P
mutant, the level of Del-5-myc
luc activity seemed to be somewhat higher than in the presence of the inactive TGFBR2E526Q
mutant, suggesting that the TGFBR2R537P
mutant has modest dominant-negative activity in this case. Total levels of TβRII receptor expression were similar across all conditions (). In aggregate, these results indicate that the TβRIIE526Q
mutant is a true loss-of-function mutant that has no dominant-negative effect on coexpressed wild-type receptor. The TβRIIR537P
mutant is equally incapable of activating classic TGFβ target genes when it is the sole receptor gene expressed (as is the case in A253 cells). On the other hand, in the presence of wild-type receptor, the TβRIIR537P
mutant seems to have retained its capability to activate some target genes (3TP-lux and SBE4-luc), whereas it is inactive with respect to Col7A1-luc and modestly dominant negative with regard to Del-5-myc
-luc. These results suggests that, in cells that are homozygous for this mutant, the classic TGFβ-regulated gene expression program is eliminated, whereas in the heterozygous situation the TGFβ-regulated gene expression program is likely to be qualitatively altered, giving rise to complex changes in the cellular phenotype.