**3.1. CNP signaling pathway**

Natriuretic peptides are one of the main classes of cGMP inducers which are known as atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP). Natriuretic peptides are secreted proteins that control cell behavior through activation of two major transmembrane receptors, natriuretic peptide receptor 1 and 2 (NPR1 and NPR2) [48–50]. Receptors NPR1 and NPR2 have guanylyl cyclase activity and synthesize cGMP in response to ligand binding. ANP and BNP signal mainly through NPR1/GC-A, while CNP predominantly activates NPR2/GC-B. All three ligands also bind to a third receptor, NPR3, which is known as the clearance receptor that does not have an intracellular signaling part from the molecule and limits ligand availability once attached and limits natriuretic peptide signaling.

Once CNP gene and its signaling were disrupted in mice, those mice suffered from postnatal dwarfism [51]. While CNP knock-out mice developed normal membranous ossification, endochondral ossification was severely impaired and all long bones and vertebrae were significantly shorter. About 70% of null mice die in the first 100 days after birth. When crossed with transgenic mice that overexpressed CNP in cartilage, knock-out mice phenotype was completely rescued.

Further organ culture experiments (femur) confirmed CNP's effect as the potent stimulator of endochondral bone growth. Other mice models were present in which Npr2 was knocked out and similar dwarf phenotype was observed, thus further confirming the importance of CNP signaling pathways. A loss-of-function mutation in the cGMP-dependent protein kinase II (cGKII gene) has also recently been identified as the cause of dwarfism in mice which is a downstream effector of CNP. When Npr3 is knocked out, an opposite phenotype of skeletal over growth is observed [52, 53].

All natriuretic peptides (ANP, BNP, and CNP) have the ability to bind NPRA, NPRB, and NPRC, while CNP has the most affinity to bind NPRB which seems to control endochondral bone growth the most. Mice deficient for ANP, BNP, or NPR1 genes were reported not to develop dwarfism or abnormal skeletal phenotypes [54, 55], suggesting that these genes play only minor supportive roles during endochondral bone formation. Overexpression of BNP in transgenic mice developed skeletal overgrowth, but this was explained as overstimulation of NPR2 by excess levels of BNP [56, 57].
