*3.8. Compensatory Proliferation*

Compensatory proliferation is a common reparative response to tissue damage that typically leads to replacement of dead cells following an injury. In irradiated murine salivary glands, induction of proliferation, as measured by increased expression of proliferating cell nuclear antigen (PCNA) or pKi67, is observed as early as 48 h post-IR [47,72] and continues through chronic time points (i.e., 30–90 days) [62,65,66,74]. Despite the increase in cell number, salivary glands remain non-functional post-IR, which correlates with reduced levels of salivary amylase, a marker of differentiated acinar cells, suggesting that the newly generated cells are maintained in an undifferentiated state [56,65,66]. Ectodysplasin A-1 receptor (EDAR) signaling typically occurs during embryogenesis to allow for fetal development of ectodermal tissues, such as skin, hair and exocrine glands, including salivary glands. Interestingly, activating this pathway with an EDAR-agonist monoclonal antibody restores salivary gland function post-IR, which correlates with a reduction in compensatory proliferation and increased levels of salivary amylase at days 30–90 [65]. The mammalian target of rapamycin (mTOR) is a critical signaling mediator that controls cell metabolism, growth, proliferation and survival, which is inhibited by rapamycin. Treating mice with the rapamycin analog, CCI-779, reduces proliferation rates while increasing levels of amylase and saliva flow rates at day 30 post-IR [88]. Likewise, post-IR IGF-1 treatment reduces the number of proliferating cells and enhances amylase levels and saliva secretion from days 9–90 post-IR [66].

Compensatory proliferation has been shown to be mediated by reduced activation of the apical polarity regulator, PKCζ, which leads to increased Jun kinase (JNK) signaling in parotid glands following IR [62]. The reduction in PKCζ activity is observed in a subset of stem and progenitor cells, as well as the entire acinar compartment at 5–30 days post-IR, which correlates with increased Ki67 levels [55,62]. Notably, mice lacking PKCζ have increased baseline proliferation rates that are unchanged post-IR and cannot be modulated by IGF-1 treatment [55,62]. Additionally, mice deficient in PKCζ and treated with IGF-1 do not show improvements in salivary gland function post-IR [55]. Together, these data illustrate the importance of the regulation of cell polarity and proliferation by PKCζ and its alteration due to the IR-induced damage response in parotid glands. Modulating proliferation downstream of PKCζ signaling may provide novel drug targets to preserve salivary gland function post-IR.
