Mkl1 is a member of the myocardin related transcription NSC-330507 factor family and a well known transcriptional co activator of serum response factor. SRF target genes, which are regulated upon recruitment of MRTF cofactors, en code proteins involved in actin cytoskeletal function that can either be structural or re lated to actin dynamics. However, Mkl1 mediated stretch induced tenascin C expression in fibroblasts did not require SRF, but instead depended on the potential DNA binding SAP domain of Mkl1. This implies a novel mode of Mkl1 action as a bona fide transcription factor Inhibitors,Modulators,Libraries in mechanotransduction. Interestingly, normal and transformed mouse mammary epithelial cells also ap pear to be highly sensitive to Mkl1 signaling, respond ing to Mkl1 overexpression with several fold induction of tenascin C.

The present study was designed to find SAP dependent Mkl1 target genes co regulated with tenascin C and to analyze whether such genes could be indicative of specific physiological Inhibitors,Modulators,Libraries states of cells that might be controlled by mechanotransduction. For our study, we made use of the HC11 mammary epithelial cell line. HC11 cells are capable of both self renewal and differentiation and can be cul tured for unlimited time in an undifferentiated state, the condition we used in our study. HC11 cells can recon stitute the ductal epithelium of a cleared mammary fat pad in vivo with ductal, alveolar and myoepithelial cells, illustrating their stem cell abilities. In addition, HC11 cells contain a mutated p53 gene that not only in creases the replicative potential of stem cells but confers predisposition to mammary carcinoma.

Undifferen tiated HC11 cells share transcriptome signatures Inhibitors,Modulators,Libraries with human breast cancer, supporting the relevance of this model for breast Inhibitors,Modulators,Libraries cancer related studies. We there fore concluded our study by investigating whether the genes co regulated with tenascin C would also be impli cated in breast cancer progression. Results Screen for SAP dependent Mkl1 target genes We devised a screening method to identify genes co regulated with tenascin C by Mkl1 in a SAP domain dependent manner without involvement of SRF. For this purpose, we used HC11 mammary epithelial cells that react strongly to the overexpression of Mkl1 with in duction of tenascin C expression.

We compared three HC11 strains that either overexpress the C terminal red fluorescent protein tagged full length Mkl1, Mkl1 RFP with a mutated SRF interaction site or Mkl1 RFP with a deletion of the SAP domain. None of the three Mkl1 variants appear to be toxic to the cells, as we have not observed Inhibitors,Modulators,Libraries any changes in viability or cell morphology. HC11 FL cells were shown to overexpress sellekchem Mkl1 7. 1 fold above the en dogenous Mkl1 present in parental HC11 cells, and were used as control cells in our study. All cell strains were FACS sorted to express similar levels of Mkl1 RFP proteins.