Further characterization of the hepatic fibrosis using relevant biological markers revealed that AhR-null liver had a marked alteration of their perivascular structure where overproduction of collagen proteins disrupts the normal hepatic histology. The damaged area contained an elevated number of fibroblasts as can be inferred from the strong staining obtained Erlotinib HCl for the fibroblast marker proteins ��-actin, fibronectin and vimentin. These results strongly demonstrate that the AhR appears to be directly involved in the development of liver fibrosis in this animal model. Several lines of evidence suggest that the AhR is also involved in TGF-�� and LTBP-1 signalling. Recent results from our group show that conditioned media from AhR-null MEFs contained two times more total TGF-�� and almost four times more active TGF-�� than AhR+/+ MEFs (Santiago-Josefat et al.

2004). In addition, using differential display, we have shown that the AhR negatively regulates LTBP-1 gene expression because null AhR?/? fibroblasts overexpressed both LTBP-1 mRNA and protein, and more significantly, overproduction of LTBP-1 appeared to contribute to TGF-�� activity (Santiago-Josefat et al. 2004). These results prompted us to analyse LTBP-1 expression in the areas of portal fibrosis in AhR-null mice livers. The results presented here show that LTBP-1 is overexpressed and colocalizes in AhR?/? liver with other fibrosis-related markers such as collagen, ��-actin and vimentin. These observations further support the role of the AhR in the development of liver fibrosis.

However, TGF-�� mRNA expression, rather than localizing exclusively to cells in the portal areas where fibrosis and LTBP-1 expression were found, was more widely distributed through the parenchyma in livers of either genotype. Thus, although TGF-�� protein levels in AhR-null mice livers were increased with respect to wild-type animals, there was no significant difference in terms of TGF-�� mRNA content. The lack of colocalization between LTBP-1 and TGF-�� mRNAs in the portal areas of AhR?/? livers suggests that a fraction of the secreted LTBP-1 might not be bound to TGF-��. In support of this observation, previous studies have reported that the fraction of LTBP-1 that is secreted devoid of TGF-�� reaches up to 90% in osteoblast cells (Dallas et al. 2002). In addition, these data also agree with that showing that AhR-null MEFs secreted increased levels of active and total TGF-�� in the absence of changes in TGF-�� mRNA expression (Santiago-Josefat et al. 2004). Taken together, these data suggest that the AhR does not regulate TGF-�� gene expression at the transcriptional level, and that TGF-�� activity could be modulated by the AhR at the AV-951 post-transductional level.