Impact of Reck expression and promoter activity in neuronal in vitro differentiation.

Reck (REversion-inducing Cysteine-rich protein with Kazal motifs) tumor suppressor gene encodes a multifunctional glycoprotein which inhibits the activity of several matrix metalloproteinases (MMPs), and has the ability to modulate the Notch and canonical Wnt pathways. Reck-deficient neuro-progenitor cells undergo precocious differentiation; however, modulation of Reck expression during progression of the neuronal differentiation process is yet to be characterized. In the present study, we demonstrate that Reck expression levels are increased during in vitro neuronal differentiation of PC12 pheochromocytoma cells and P19 murine teratocarcinoma cells and characterize mouse Reck promoter activity during this process. Increased Reck promoter activity was found upon induction of differentiation in PC12 cells, in accordance with its increased mRNA expression levels in mouse in vitro models. Interestingly, Reck overexpression, prior to the beginning of the differentiation protocol, led to diminished efficiency of the neuronal differentiation process. Taken together, our findings suggest that increased Reck expression at early stages of differentiation diminishes the number of neuron-like cells, which are positive for the beta-3 tubulin marker. Our data highlight the importance of Reck expression evaluation to optimize in vitro neuronal differentiation protocols.

Extracellular matrix dynamics during mesenchymal stem cells differentiation.

Mesenchymal stem cells (MSCs) are stromal cells that display self-renewal and multipotent differentiation capacity. The repertoire of mature cells generated ranges but is not restricted to: fat, bone and cartilage. Their potential importance for both cell therapy and maintenance of in vivo homeostasis is indisputable. Nonetheless, both their in vivo identity and use in cell therapy remain elusive. A drawback generated by this fact is that little is known about the MSC niche and how it impacts differentiation and homeostasis maintenance. Hence, the roles played by the extracellular matrix (ECM) and its main regulators namely: the Matrix Metalloproteinases (MMPs) and their counteracting inhibitors (TIMPs and RECK) upon stem cells differentiation are only now beginning to be unveiled. Here, we will focus on mesenchymal stem cells and review the main mechanisms involved in adipo, chondro and osteogenesis, discussing how the extracellular matrix can impact not only lineage commitment, but, also, their survival and potentiality. This review critically analyzes recent work in the field in an effort towards a better understanding of the roles of Matrix Metalloproteinases and their inhibitors in the above-cited events.

Isolation and characterization of novel RECK tumor suppressor gene splice variants.

Glioblastoma multiforme is the most common and lethal of the central nervous system glial-derived tumors. RECK suppresses tumor invasion by negatively regulating at least three members of the matrix metalloproteinase family: MMP-9, MMP-2, and MT1-MMP. A positive correlation has been observed between the abundance of RECK expression in tumor samples and a more favorable prognosis for patients with several types of tumors. In the present study, novel alternatively spliced variants of the RECK gene: RECK-B and RECK-I were isolated by RT-PCR and sequenced. The expression levels and profiles of these alternative RECK transcripts, as well as canonical RECK were determined in tissue samples of malignant astrocytomas of different grades and in a normal tissue RNA panel by qRT-PCR. Our results show that higher canonical RECK expression, accompanied by a higher canonical to alternative transcript expression ratio, positively correlates with higher overall survival rate after chemotherapeutic treatment of GBM patients. U87MG and T98G cells over-expressing the RECK-B alternative variant display higher anchorage-independent clonal growth and do not display modulation of, respectively, MMP-2 and MMP-9 expression. Our findings suggest that RECK transcript variants might have opposite roles in GBM biology and the ratio of their expression levels may be informative for the prognostic outcome of GBM patients.