Our study defines for the first time distinct functional roles for Smad2 and Smad3 in postnatal liver. We report that postnatal development and homeostasis are not perturbed in mice with hepatocyte-specific deletion of Smad2 or a double knockout of Smad2 and Smad3. Thus, R-Smads are not required to maintain normal physiological conditions in hepatocytes. These findings are consistent with a recent report demonstrating normal development and appearance of liver in mice with Cre/loxP mediated hepatocyte-specific knockout of TgfbR2 (36
), indicating that TGF-β signaling is dispensable in hepatocytes under unchallenged conditions in vivo. In addition, it is possible that Smad2 and Smad3 deficiency in hepatocytes may be compensated for under physiological conditions by unspecified stromal-epithelial interactions.
In contrast, our studies demonstrate distinct functional roles of Smad2 and Smad3 in hepatocytes when the physiological context is challenged in vivo. We identify a novel functional role for Smad2 as a cell-autonomous negative regulator of hepatocyte growth in vitro and in vivo. This conclusion is supported by significantly increased baseline rates of proliferation of primary Smad2-deficient hepatocytes compared to wild-type and Smad3-deficient hepatocytes in vitro and by significantly and persistently increased growth rates of transplanted Smad2-deficient hepatocytes for up to 3 months in a liver cell transplantation model in mice. These results suggest that basal proliferation of Smad2-deficient hepatocytes is reset to permit increased proliferative rates compared to wild-type control cells and that the reset is likely independent of TGF-β activity. The increased proliferation was associated with a strikingly increased abundance of cyclin D1. Cyclin D1 promotes G1
/S progression of cell cycle (26
), and its rapid increase in regenerating liver after partial hepatectomy was increased in hepatocyte-specific Tgfbr2 knockout livers compared to WT (30
). Interestingly, TGF-β completely suppressed aberrant cyclin D1 expression in S2HeKO, indicating that the increased basal level of cyclin D1 is independent of extracellular TGF-β.
Our results further demonstrate that Smad2 mediates negative control of hepatocyte proliferation after toxic liver injury induced by single necrogenic dose of CCl4 (52
). This conclusion is supported by significantly increased hepatocyte proliferation in livers of S2HeKO
mice compared to control and Smad3KO
mice. It remains to be determined whether this function is TGF-β dependent or independent. However, a recent report demonstrated that nuclear localization of Smad2 was reduced by 50% in Albcre/Tgfbr2f/f
livers compared to controls after partial hepatectomy (36
), indicating that activation of Smad2 may be dependent on TGF-β signaling in a different model of regeneration. Our results confirm an antimitogenic role for Smad2 in liver regeneration after acute toxic injury, whereas we were unable to demonstrate a role for Smad3. We also observed that both long Smad2FL and short Smad2Δexon3 transcripts are present in this model (data not shown). It will be important to determine which of these splice forms mediates the antimitogenic activity, since short Smad2Δexon3 has been shown to be capable of mediating all TGF-β signaling during embryonic development, whereas long Smad2FL was neither essential nor sufficient for TGF-β signaling or embryonic development (12
In contrast to our in vivo observations, our in vitro studies demonstrate that Smad3 is required for TGF-β-stimulated growth inhibition of primary hepatocyte, whereas Smad2 was not required. However, the antimitogenic role of Smad3 appears to be highly cell type and/or context dependent. For example, Smad3 is not required for TGF-β growth inhibition in mammary gland epithelial cells (51
) but is required in ras-transformed keratinocytes (43
). Overexpression of Smad2, but not of Smad3, significantly reduced growth of Mv1Lu mink lung epithelial cells xenografted in SCID mice (39
). Moreover, epidermal keratinocyte proliferation was reduced in 12-tetradecanoyl phorbol myristate acetate-induced skin of Smad3-deficient mice compared to controls (19
). Together our in vivo and in vitro results and these published observations indicate that a role of Smad3 as a mediator of TGF-β-induced epithelial growth inhibition may not be universally applicable and appears to be highly context dependent.
Surprisingly, Smad2-deficient hepatocytes in primary culture were unable to maintain a differentiated epithelial phenotype and spontaneously manifested morphological and molecular features characteristic of EMT similar to those induced by TGF-β in control hepatocytes. In contrast, untreated S3KO cells had characteristic differentiated epithelial cell features, and TGF-β was unable to induce EMT in these cells, indicating that Smad3 was required for TGF-β induced EMT in vitro, as previously reported (37
). Although previous studies using overexpression or dominant-negative interference systems indicated that Smad2 and Smad3 mediate TGF-β-induced EMT (41
), the results from our genetic approach presented here suggest that Smad2 is not involved in TGF-β-induced EMT in primary hepatocytes, whereas Smad3 is required. In fact, our results suggest that Smad2 function is required for stable epithelial phenotype of primary hepatocytes, possibly independent of TGF-β activity. However, the relevance of this surprising Smad2 function in vivo, for example, in liver injury or hepatocarcinogenesis, remains to be determined.
Our findings have implications for hepatocarcinogenesis. Genetic inactivation of Tgfbr2, Smad2, and Smad4 in hepatocellular carcinoma have been reported, albeit with various frequencies (14
). In addition, there is substantial evidence for epigenetic inactivation of Smad2 or Smad3 function in various cancers, commonly mediated by direct interaction with oncoproteins (10
). Moreover, TGF-β may exert dual roles in carcinogenesis where TGF-β switches from a tumor suppressor in the premalignant stages of tumorigenesis to a proto-oncogene function at later stages of disease leading to metastasis (35
). The molecular determinants that mediate this functional switch remain poorly understood. Our results lead us to speculate that the loss of Smad2 function (genetic or epigenetic) during carcinogenesis may increase the proliferative and metastatic potential, whereas loss of Smad3 function may decrease the metastatic potential of tumor cells. Thus, it is possible that the critical functional switch may be determined by changes of the relative balance of Smad2 (antimetastatic signaling) and Smad3 (prometastatic signaling) in malignant cells.
The present results confirm previous results obtained from mouse fibroblasts (49
) indicating that Smad2 function is not required or may negatively modulate transcriptional regulation by TGF-β if Smad3 is present. Interestingly, Smad3-deficient hepatocytes showed various degrees of partial transcriptional responsiveness to TGF-β if Smad2 is present, depending on the individual target gene. In contrast, transcriptional activity of TGF-β was universally lost in hepatocytes lacking both Smad2 and Smad3. Based on genetic isoform replacement experiments in mice, expression of the Smad2 splice form Smad2Δexon3 or Smad3 cDNAs under control of the Smad2 locus was sufficient to mediate all transcriptional responses of TGF-β required for normal development in Smad2-deficient embryos (12
). In contrast, expression of full-length Smad2 cDNA under control of the Smad2 locus was not sufficient to rescue phenotypes of Smad2 knockout embryos, indicating that the long form of Smad2 may exert functions unrelated to transcriptional regulation by TGF-β (12
). Both Smad2 isoforms are present in hepatocytes (data not shown). Thus, our results suggest that the Smad2Δexon3 may compensate partially for transcriptional function of Smad3 in S3KO hepatocytes. Moreover, Smad2/Smad3 double knockout is also associated with a striking reset (reduced) of baseline transcript levels of target genes that are activated by TGF-β, indicating that deficiency of Smad2 and Smad3 profoundly alters the basal and inducible transcriptome of hepatocytes. Interestingly, although double deficiency of Smad2 and Smad3 is compatible with normal hepatocyte function under physiological conditions postnatally, DKO hepatocytes have dramatically increased susceptibility to undergo necrosis or apoptosis in response to toxic liver injury in vivo and to stress induced by standard hepatocyte isolation procedures.