Calcium signaling : molecular mechanisms and cellular consequences

Cells exploit calcium (Ca2+) signaling to transmit information. In a multicellular organism, each cell must be able recognize, process, and respond to information received from the surrounding environment. In this thesis I investigate molecular mechanisms and cellular consequences of Ca2+ signaling.

Ouabain is an endogenous hormone, and ligand of the Na,K-ATPase, that has previously been shown to induce Ca2+ oscillations in renal cells. Here we report that the N-terminal tail of the Na,K-ATPase alpha-subunit binds directly to the N-terminus of the inositol 1,4,5-trisphosphate receptor (InsP3R). Three amino acid residues in the Na,K-ATPase N-terminal tail, LKK, conserved in most species, are essential for this binding to occur. Over-expression of a peptide encoding for the N-terminal tail impaired ouabain-triggered Ca2+ oscillations. Thus we have identified a well conserved Na,K-ATPase-motif that binds to the InsP3R and is vital for intracellular Ca2+ signaling. The role of Na,K-ATPase signaling during dendritogenesis was examined by treating embryonic cortical rat neurons with ouabain. We report that Na,K-ATPase signal transduction triggers dendritic growth as well as a transcriptional program dependent on CREB and CRE-mediated gene expression, primarily regulated via Ca2+/calmodulin-dependent protein kinases. This signaling cascade also involves intracellular Ca2+ oscillations and sustained phosphorylation of mitogen-activated protein kinases. These results suggest a novel role for the Na,K-ATPase as a modulator of dendritic growth in developing neurons.

We explored the Ca2+ signaling properties of differentiating mouse embryonic stem cells. Spontaneous Ca2+ activity was shown to be present in neural progenitors derived from mouse embryonic stem cells. This Ca2+ activity was dependent on influx of extracellular Ca2+ through plasma membrane channels, since removal of extracellular Ca2+ from the medium and inhibition of voltage-dependent channels blocked the signaling event. Cross-correlation analysis revealed that the spontaneous Ca2+ activity was more synchronous in sub-populations of the neural progenitors than in the undifferentiated mouse embryonic stem cells. A significant reduction Ca2+ activity was observed when cells were challenged with gap junction blockers. Inhibiting the spontaneous Ca2+ activity significantly reduced the number of dividing cells.

In conclusion, this thesis presents novel data on a conserved Na,K-ATPasemotif important for the N-terminal signaling activity and demonstrates a role of Na,K-ATPase in dendritic growth in developing cortical neurons. Further, spontaneous Ca2+ activity in neural progenitors derived from embryonic stem cells is dependent on extracellular Ca2+ and is important for cell division.

List of scientific papers

I. Zhang S, Malmersjö S, Li J, Ando H, Aizman O, Uhlén P, Mikoshiba K, Aperia A (2006). Distinct role of the N-terminal tail of the Na,K-ATPase catalytic subunit as a signal transducer. J Biol Chem. 281(31): 21954-62. Epub 2006 May 24
https://pubmed.ncbi.nlm.nih.gov/16723354

II. Desfrere L, Karlsson M, Hiyoshi H, Malmersjö S, Nanou E, Estrada M, Miyakawa A, Lagercrantz H, El Manira A, Lal M, Uhlén P (2008). Na,K-ATPase signal transduction triggers CREB activation and dendritic growth. [Submitted]
https://pubmed.ncbi.nlm.nih.gov/19164762

III. Edman LC, Mira H, Erices A, Malmersjö S, Andersson E, Uhlén P, Arenas E (2008). Alpha-chemokines regulate proliferation, neurogenesis, and dopaminergic differentiation of ventral midbrain precursors and neurospheres. Stem Cells. 26(7): 1891-900. Epub 2008 Apr 24
https://pubmed.ncbi.nlm.nih.gov/18436867

IV. Malmersjö S, Rebellato P, Liste I, Sunner H, Arenas E, Uhlén P (2008). Spontaneous calcium activity regulates proliferation in neural progenitors derived from embryonic stem cells. [Manuscript]
https://doi.org/10.1073/pnas.1220179110