The bone marrow contains hematopoietic as well as nonhematopoietic stem cells called mesenchymal stem cells (MSCs) or stromal cells. MSCs can be easily isolated and undergo extensive expansion in vitro. They are multi-potent and can give rise to nonmesenchymal cells such as hepatocytes and neural cells. Therefore, MSCs are a promising source for therapeutic treatment of a number of diseases. How MSCs is regulated to differentiate into other cell types at the transcriptional and translational level, however, is poorly understood. We intend to uncover the mechanism(s) that govern stem cell proliferation versus differentiation at the transcriptional level. Understanding how re-programming the gene expression profiles alters stem cell fate may help to guide and control cell differentiation. It has been reported that cAMP, an important intracellular second messenger, is able to induce the differentiation of MSCs into neural cells. Elevated cAMP leads to intracellular signaling that activates the CREB (cAMP response element binding protein) family proteins. Within the CREB family proteins, CREB is regulated by its phosphorylation status. Phosphorylation of the CREB serine 133 site enables it to recruit the coactivator CBP, which serves as a bridging protein to recruit the general transcription machinery. Upon recruitment of CBP, phosphorylated CREB is thereby activated and able to initiate the transcription of cAMP-responsive genes. On the other hand, another CREB family protein ICER (inducible cAMP early repressor) contains the DNA binding domain but not the transcriptional activation domain. Therefore ICER competes with CREB for binding to CRE motif but can not initiate transcription. Since ICER itself contains four consecutive CRE motifs and is also inducible by activated CREB, it forms a negative feedback loop to down-regulate its own transcription. As a consequence, cAMP-responsive genes may be regulated in a dynamic manner by the relative abundance of activated CREB and ICER. Upon the addition of cAMP inducing agents to MSCs, we have found that cAMP lowered the cell proliferation rate and concomitantly caused morphological changes of the MSCs into neural cells. MSCs incubated with forskolin and IBMX for six days expressed astrocyte marker GFAP (Glial fibrillary acidic protein) and neuron marker NF-200 (neurofilament-200). Cell proliferation assay suggests that the proliferation rate of the MSCs decreased significantly upon forskolin and IBMX treatment. In cell cycle progression, many cell cycle regulators such as cyclin D and cyclin A are dynamically regulated. Cyclin D1 associates with CDK4/6 (cyclin dependent kinase 4/6) to regulate G1/S phase transition. Activated CREB induces the transcription of cyclin D1 while ICER down-regulates the transcription of cyclin D1 by competing with CREB for binding to the CRE motif. Whether cyclin D1 is up- or down-regulated largely depends on the relative activity of CREB and ICER. Since cyclin D1 is a critical regulator of G1/S phase transition, its expression influences cell cycle progression and consequently stem cell proliferation. Delayed G1/S phase slows stem cell self-renewal and possibly prepares the stem cells for differentiation.

Extended Abstract Status: Not Uploaded