INTRACELLULAR SIGNALING BY BINDING-SITES FOR THE ANTIPSORIATIC AGENT MONOMETHYLFUMARATE ON HUMAN GRANULOCYTES

Monomethylfumarate (MMF), the most active metabolite of the new antips oriasis drug Fumaderm(R), stimulates an anti-inflammatory mediator pro file in human leucocytes and inhibits the proliferation of keratinocyt es, These effects of MMF on cells in vitro may in part explain the ben eficial action of Fumaderm(R) in patients. In addition, we have report ed that MMF stimulates an increase in the intracellular free Ca2+ conc entration ([Ca2+](i)) and the cyclic adenosine monophosphate (cAMP) co ncentration in granulocytes and keratinocytes. Because Ca2+ and cAMP c ontrol many physiological cellular responses, including cell prolifera tion and inflammatory mediator production, the present study focused o n the intracellular signal transduction pathway which links interactio n between MMF and granulocytes with increases in [Ca2+](i) and the cAM P concentration. The increase in [Ca2+](i) in granulocytes after MMF d epended both on extracellular Ca2+ and Ca2+ from intracellular stores. Ca2+ is essential for the increase in the cAMP concentration after st imulation with MMF. The results found for pharmacological inhibitors o f various protein kinases suggest that a staurosporine-sensitive prote in kinase different from protein kinase C (PKC) and protein kinase A i s involved in the MMF-induced increase in [Ca2+](i) in granulocytes. A s MMF activated protein tyrosine kinase (PTK), and inhibition of this protein kinase partially reduced the increase in [Ca2+](i) in granuloc ytes, PTK activity most likely is involved, In addition, activation of protein kinase histone 4 (PKH4) probably plays a part in the MMF-stim ulated increase in [Ca2+](i) in granulocytes as well. As MMF stimulate d an increase in the GTP-ase activity of membranes and pertussis toxin (PTX) inhibited the increase in the [Ca2+](i) and PKH4 activity of gr anulocytes stimulated by this compound, it is concluded that MMF activ ates PTX-sensitive G proteins. Competition binding studies with radiol abelled dimethylfumarate (DMF) and unlabelled DMF and MMF revealed the presence of specific binding sites for methylated fumarates on granul ocytes. In summary, MMF binds to specific sites on the plasma membrane of cells, This interaction activates pertussis toxin-sensitive G prot eins which then stimulate an increase in PTK and PKH4 activity. These protein kinases may regulate the rise in [Ca2+](i) and the intracellul ar cAMP concentration. Elevated [Ca2+](i) and intracellular cAMP conce ntration stimulate protein kinases that regulate transcription factors , Activation of these factors results in induction of downstream gene expression and thus controls cell functions, e.g. cell proliferation a nd production of inflammatory mediators, as has been found for cells i ncubated with MMF.