Induction of colitis and rapid development of colorectal tumors in mice deficient in the neuropeptide PACAP.

Pituitary adenylyl cyclase activating peptide (PACAP) is expressed in central, sensory, autonomic, and enteric neurons. Although it classically acts as a neurotransmitter/neuromodulator, recent studies indicate that PACAP can also regulate immune function. To this effect, PACAP has been shown to reduce clinical symptoms and inflammation in mouse models of human immune-based diseases such as rheumatoid arthritis, Crohn's Disease, septic shock and multiple sclerosis. Despite these findings, the role of the endogenous peptide in regulating immune function is unknown. To determine if endogenous PACAP plays a protective role in inflammatory bowel disease (IBD) and IBD-associated colorectal cancer in mice, PACAP-deficient (KO) mice were subjected to 3 cycles of dextran sulfate sodium (DSS) in drinking water over 2 months, an established mouse model for colitis. Compared to wild type (WT) controls, PACAP KO mice exhibited more severe clinical symptoms of colitis and had significantly higher colonic inflammation on pathological examination. Moreover, 60% of the PACAP KO mice developed colorectal tumors with an aggressive-appearing pathology. Consistent with published data, DSS-treated WT mice did not develop such tumors. The results demonstrate a new mouse model which rapidly develops inflammation-associated colorectal cancer in the absence of a carcinogen.

Disruption of the PACAP gene promotes medulloblastoma in ptc1 mutant mice.

Hedgehog (Hh) proteins and cAMP-dependent protein kinase A (PKA) generally play opposing roles in developmental patterning events. Humans and mice heterozygous for mutations in the sonic hedgehog (Shh) receptor gene patched-1 (ptc1) have an increased incidence of certain types of cancer, including medulloblastoma (MB), a highly aggressive tumor of the cerebellum. Despite the importance of PKA in Hh signaling, little is known about how PKA activity is regulated in the context of Hh signaling, or the consequences of improper regulation. One molecule that can influence PKA activity is pituitary adenylyl cyclase-activating peptide (PACAP), which has been shown to regulate cerebellar granule precursor proliferation in vitro, a cell population thought to give rise to MB. To test for a PACAP/Hh interaction in the initiation or propagation of these tumors, we introduced a PACAP mutation into ptc1 mutant mice. Deletion of a single copy of PACAP increased MB incidence approximate 2.5-fold, to 66%, thereby demonstrating that PACAP exerts a powerful inhibitory action on the induction, growth or survival of these tumors. Tumors from PACAP/ptc1 mutant mice retained PACAP receptor gene expression, and exhibited superinduction of Hh target genes compared to those from ptc1+/- mice. Moreover, PACAP inhibited proliferation of cell lines derived from tumors in a PKA-dependent manner, and inhibited expression of the Hh target gene gli1. The results provide genetic evidence that PACAP acts as a physiological factor that regulates the pathogenesis of Hh pathway-associated MB tumors.

Impairment of axotomy-induced pituitary adenylyl cyclase-activating peptide gene expression in T helper 2 lymphocyte-deficient mice.

CD4+ (T helper) lymphocytes appear to play important roles in neuron survival and regeneration after injury, although their functions in regulating gene expression in injured neurons are unknown. Mice with targeted mutations in the STAT4 and STAT6 genes are deficient in T helper (Th)1 and Th2 responses, respectively, and have been used to determine the relative importance of T helper subsets in a variety of inflammatory processes. As pituitary adenylyl cyclase-activating peptide mRNA is normally strongly induced in facial motor neurons after axotomy, we examined this induction in Th1 and Th2 lymphocyte-deficient and control Balb/C wild-type mice. As previously reported, pituitary adenylyl cyclase-activating peptide gene expression was strongly induced in ipsilateral but not contralateral motor neurons in the facial motor nucleus of wild-type mice. The mean number of hybridizing motor neurons in STAT4-deficient mice did not differ from that in wild-type mice, whereas the number in STAT6 mice was reduced by more than 50%. The results indicate that STAT6 plays a key role in the upregulation of pituitary adenylyl cyclase-activating peptide gene expression in facial motor neurons after injury, possibly through its role in regulating T helper cell differentiation to the type 2 phenotype.

Restoration of axotomy-induced PACAP gene induction in SCID mice with CD4+ T-lymphocytes.

PACAP is a neuropeptide with putative neuroprotective, regenerative, and immunomodulatory actions. PACAP mRNA is up-regulated in motor neurons following facial nerve axotomy in wild type, but not immunodeficient SCID mice. Because CD4+ lymphocytes appear to be neuroprotective in facial nerve and other injury models, we studied PACAP gene expression in SCID mice preinfused with CD4+ enriched splenocytes. Whereas the mean number of PACAP hybridizing neurons after axotomy was reduced by 75% in uninfused SCID mice, infusion of CD4+ enriched splenocytes restored the number to a value not significantly different than controls. The CD4+ cell-dependent induction of PACAP in motor neurons may thus be a factor in the cascade of events triggered by immune cells that ultimately lead to nerve regeneration.