Identification of receptors and signaling pathways for orphan bone morphogenetic protein/growth differentiation factor ligands based on genomic analyses.

There are more than 30 human transforming growth factor beta/bone morphogenetic protein/growth differentiation factor (TGFbeta/BMP/GDF)-related ligands known to be important during embryonic development, organogenesis, bone formation, reproduction, and other physiological processes. Although select TGFbeta/BMP/GDF proteins were found to interact with type II and type I serine/threonine receptors to activate downstream Smad and other proteins, the receptors and signaling pathways for one-third of these TGFbeta/BMP/GDF paralogs are still unclear. Based on a genomic analysis of the entire repertoire of TGFbeta/BMP/GDF ligands and serine/threonine kinase receptors, we tested the ability of three orphan BMP/GDF ligands to activate a limited number of phylogenetically related receptors. We characterized the dimeric nature of recombinant GDF6 (also known as BMP13), GDF7 (also known as BMP12), and BMP10. We demonstrated their bioactivities based on the activation of Smad1/5/8-, but not Smad2/3-, responsive promoter constructs in the MC3T3 cell line. Furthermore, we showed their ability to induce the phosphorylation of Smad1, but not Smad2, in these cells. In COS7 cells transfected with the seven known type I receptors, overexpression of ALK3 or ALK6 conferred ligand signaling by GDF6, GDF7, and BMP10. In contrast, transfection of MC3T3 cells with ALK3 small hairpin RNA suppressed Smad signaling induced by all three ligands. Based on the coevolution of ligands and receptors, we also tested the role of BMPRII and ActRIIA as the type II receptor candidates for the three orphan ligands. We found that transfection of small hairpin RNA for BMPRII and ActRIIA in MC3T3 cells suppressed the signaling of GDF6, GDF7, and BMP10. Thus, the present approach provides a genomic paradigm for matching paralogous polypeptide ligands with a limited number of evolutionarily related receptors capable of activating specific downstream Smad proteins.

Mustard oil has differential effects on the response of trigeminal caudalis neurons to heat and acidity.

Topical application of mustard oil (allyl isothiocyanate) to the skin or injection into joints induces hyperalgesia, allodynia, and neuroinflammation. However, when applied to the oral or nasal mucosa, mustard oil evokes a desensitizing pattern of irritation. Presently we investigated the responses of neurons in superficial laminae of trigeminal subnucleus caudalis (Vc) to noxious thermal (53 degrees C) and chemical (pentanoic acid; 200 mM) stimuli prior to and following lingual mustard oil application. A low concentration of mustard oil (0.125%) applied by constant flow (0.5 ml/min; 15 min), initially excited Vc neurons followed by partial desensitization. Responses to noxious heat were unchanged following mustard oil. A high concentration of mustard oil (1.25%) initially excited Vc neurons followed quickly (within 20 s) by nearly complete desensitization. The desensitization was transient since reapplication of mustard oil approximately 20 min later elicited a comparable response that also rapidly desensitized. Mustard oil also transiently cross-desensitized Vc responses to pentanoic acid (to 52%), in striking contrast to noxious heat-evoked responses which were significantly sensitized to approximately 160% of pre-mustard oil levels. The data suggest that the effect of mustard oil on subsequent lingual nociceptive responses is concentration dependent, transient, and modality specific.