Hyaluronan esters drive Smad gene expression and signaling enhancing cardiogenesis in mouse embryonic and human mesenchymal stem cells

• Published in: PloS one 2010-11, Vol.5 (11), p.e15151
• Main Authors: Maioli, Margherita, Santaniello, Sara, Montella, Andrea, Bandiera, Pasquale, Cantoni, Silvia, Cavallini, Claudia, Bianchi, Francesca, Lionetti, Vincenzo, Rizzolio, Flavio, Marchesi, Irene, Bagella, Luigi, Ventura, Carlo
• Format: Article
• Language: English
• Subjects: Acids; Actinin; Analysis; Animals; Biology; Biotechnology; Blotting, Western; Butyric Acid - pharmacology; Cancer; Cardiomyocytes; Cell Differentiation - drug effects; Cell fate; Cells, Cultured; Chemistry; Chromatin; Combinatorial analysis; Conductors; Drosophila; Embryo cells; Embryogenesis; Embryonic Stem Cells - cytology; Embryonic Stem Cells - metabolism; Embryos; Engineering; Esters; Fetuses; Fluorescence; Gene expression; Gene Expression Regulation - drug effects; Gene transfer; Genes; Genetic research; Growth factors; Heart; HEK293 Cells; Humans; Hyaluronic acid; Hyaluronic Acid - chemistry; Hyaluronic Acid - pharmacology; Immunofluorescence; Insects; Kinases; Laboratories; Mathematical models; Medical research; Medicine; Membranes; Mesenchymal stem cells; Mesenchymal Stromal Cells - cytology; Mesenchymal Stromal Cells - metabolism; Mesenchyme; Mice; Molecular biology; Myocytes, Cardiac - cytology; Myocytes, Cardiac - metabolism; Nkx2.5 protein; Nuclei; Pattern formation; Placenta; Polymerase chain reaction; Precipitation (Meteorology); Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Rodents; Runoff; Saturated fatty acids; Signal Transduction - drug effects; Signaling; Smad protein; Smad Proteins - genetics; Smad Proteins - metabolism; Smad1 Protein - genetics; Smad1 Protein - metabolism; Smad3 Protein - genetics; Smad3 Protein - metabolism; Smad4 protein; Smad4 Protein - genetics; Smad4 Protein - metabolism; Smad7 protein; Smad7 Protein - genetics; Smad7 Protein - metabolism; Stem cell transplantation; Stem cells; Studies; Transcription; Transcription (Genetics); Transgenic animals; Tretinoin - pharmacology
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0015151

Development of molecules chemically modifying the expression of crucial orchestrator(s) of stem cell commitment may have significant biomedical impact. We have recently developed hyaluronan mixed esters of butyric and retinoic acids (HBR), turning cardiovascular stem cell fate into a high-yield process. The HBR mechanism(s) remain still largely undefined. We show that in both mouse embryonic stem (ES) cells and human mesenchymal stem cells from fetal membranes of term placenta (FMhMSCs), HBR differentially affected the patterning of Smad proteins, one of the major conductors of stem cell cardiogenesis. Real-time RT-PCR and Western blot analyses revealed that in both cell types HBR enhanced gene and protein expression of Smad1,3, and 4, while down-regulating Smad7. HBR acted at the transcriptional level, as shown by nuclear run-off experiments in isolated nuclei. Immunofluorescence analysis indicated that HBR increased the fluorescent staining for Smad1,3, and 4, confirming that the transcriptional action of HBR encompassed the upregulation of the encoded Smad proteins. Chromatin immune precipitation and transcriptional analyses showed that HBR increased the transcription of the cardiogenic gene Nkx-2.5 through Smad4 binding to its own consensus Smad site. Treatment of mouse ES cells and FMhMSCs with HBR led to the concomitant overexpression of both Smad4 and α-sarcomeric actinin. Smad4 silencing by the aid of lentiviral-mediated Smad4 shRNA confirmed a dominant role of Smad4 in HBR-induced cardiogenesis. The use of HBR may pave the way to novel combinatorial strategies of molecular and stem cell therapy based on fine tuning of targeted Smad transciption and signaling leading to a high-throughput of cardiogenesis without the needs of gene transfer technologies.