During endochondral development, elongation of the bone collar occurs coordinately
with growth of the underlying cartilaginous growth plate. Transglutaminases (TGases)
are upregulated in hypertrophic chondrocytes, and correlative evidence suggests a
relationship between these enzymes and mineralization. To examine whether TGases are
involved in regulating mineralization/osteogenesis during bone development, we devised
a coculture system in which one cellular component (characterized as preosteoblastic)
is derived from the nonmineralized region of the bone, and the other cellular component
is hypertrophic chondrocytes. In these cocultures, mineralization is extensive, with
the preosteoblasts producing the mineralized matrix, and the chondrocytes regulating
this process. Secreted regulators are involved, as conditioned medium from chondrocytes
induces mineralization in preosteoblasts, but not vice versa. One factor is TGase.
In the cocultures, inhibition of TGase reduces mineralization, and addition of the
enzyme enhances it. Exogenous TGase also induces markers of osteoblastic differentiation
(i.e., bone sialoprotein and osteocalcin) in the preosteoblasts, suggesting their
differentiation into osteoblasts. Two possible signaling pathways may be affected
by TGase and result in increased mineralization (i.e., TGF-beta and protein kinase
A pathways). Addition of exogenous TGF-beta2 to the cocultures increases mineralization;
though, when mineralization is induced by TGase, there is no detectible elevation
of TGF-beta, suggesting that these two factors stimulate osteogenesis by different
pathways. However, an interrelationship seems to exist between TGase and PKA-dependent
signaling. When mineralization of the cocultures is stimulated through the addition
of TGase, a concomitant reduction (50%) in PKA activity occurs. Consistent with this
observation, addition of an activator of PKA (cyclic AMP) to the cultures inhibits
matrix mineralization, while known inhibitors of PKA (H-89 and a peptide inhibitor)
cause an increase in mineralization. Thus, at least one mechanism of TGase stimulation
probably involves inhibition of the PKA-mediated signaling.