Altered signalling pathways of bone formation in PACAP KO mice

Pituitary adenylate cyclase activating polypeptide (PACAP) is a naturally secreted signaling peptide proven to have important regulatory roles in the differentiation of the CNS as well as in several peripheral tissues. However, its role in the signaling pathways of osteogenesis and bone regeneration is a field that is still under investigation. In our research we have been monitoring the morphology of the diaphysis of femur in PACAP KO and WT C57BL6 mice while following the signaling pathways that regulate osteogenesis.
With whole limb Alizarin staining and CT, the anterior cortical bone of PACAP KO mice was shown to be significantly thicker than that of the WT mice. In addition, the organic ECM content of the genetically modified mice e.g. collagen type I was increased in the PACAP KO. Expression of PAC1 and VPAC2 receptors was demonstrated, which activation may have an influence on the PKA signaling pathway. The classical downstream pathway of PKA is via phosphorylation of CREB, but it was shown to have decreased expression. However increased Runx2 was detected in PACAP KO. Signs of enhanced bone formation, such as increased protein expression of ALP, osterix, osteocalcin and osteopontin were detected with Western blot. Elements of BMP signaling pathways of PACAP KO were also investigated and were BMP 6 and 7 showing elevated expression along with increased expression and nuclear presence of Smad1. Moreover, the PACAP KO mice also showed increased expression of IHH, SHH and Gli1, all of these being elements of the Hedgehog signaling pathway.
Our results indicate that PACAP KO mice show various signs of disturbed osteogenesis. Clarifying whether the absence of PACAP itself or activation of any compensatory mechanisms are causative in this phenomenon requires further experiments.