The results of this study demonstrate the effect of stress on a number of components of the CRH family in rat adrenal medulla. Basal levels of their mRNAs were very distinct, with expression of Ucn2 several orders of magnitude higher than CRH, Ucn1, and CRH receptors. Ucn3 mRNA was undetectable in the rat adrenal medulla. Furthermore, we found that a single episode of immobilization stress triggers a dramatic increase in Ucn2 gene expression. With repeated stress Ucn2 mRNA levels were also elevated. Single and repeated IMO also led to an increase in CRH mRNA levels. CRHR1 gene expression was up-regulated only after single IMO. Stress had no significant effect on CRHR2 mRNA levels in rats 3 h after either 1x or 6xIMO, however a modest, but significant reduction, was observed immediately following 1xIMO in WT mice. The results from glucocorticoid-treated PC12 cells and CRH-KO mice point to the likely importance of the HPA axis and the stress-induced rise in glucocorticoids for increased Ucn2 gene expression, and perhaps also for other components of the CRH system.
Relative gene expression of CRH family members in rat adrenal medulla
Although other studies (Fukuda et al. 2005; Dermitzaki et al. 2007) have also observed the expression of various CRH family members (Ucn2, CRH, Ucn1, CRHR1, and CHRR2) in the adrenal medulla, consistent with a local CRH system, this is the first study, to our knowledge, to indicate that gene expression of Ucn2 is the most abundant of all CRH members. We also showed the ratio of mRNA abundance of individual CRH family members. Basal Ucn2 mRNA levels are about 200-fold higher than that of CRH, while Ucn1 mRNA is on the border of detection and Ucn3 mRNA is essentially absent in rat adrenal medulla. An absence of Ucn3 mRNA has also been demonstrated by in situ hybridization in human adrenal medulla (Fukuda et al. 2005). In this regard, studies in knockout animals showed a limited effect of Ucn3 deficiency, as compared to Ucn2 deficiency, on adrenal function and growth (Riester et al. 2012).
In contrast to our study, Dermitzaki et al. (2007), using immunohistochemical methods, reported similar levels of CRH, Ucn1, Ucn2, and their receptors CRHR1 and CRHR2 throughout the rat adrenal medulla. However, this is based solely on the specificity of the antibodies used. Unfortunately, to our knowledge, there are no suitable commercially available antibodies for rat Ucn2 for western blot analysis.
Stress-induced changes in gene expression of adrenomedullary CRH family members and potential functional implications
Not only is the mRNA for Ucn2 the most abundant of the CRH family members under basal conditions but it also displayed the greatest increase in mRNA levels (greater than 30-fold) with a single exposure to IMO, highlighting its likely functional significance perhaps on the local catecholaminergic system. We hypothesize that the robust IMO-induced increase in Ucn2 gene expression in rat adrenal medulla may have an autocrine/paracrine regulatory role on catecholamine secretion and biosynthesis with stress conditions. It was previously shown that Ucn2 can modulate catecholamine biosynthesis and secretion. Treatment of PC12 cells with Ucn2 stimulates NE secretion, induces phosphorylation of tyrosine hydroxylase (TH) (Nemoto et al. 2005), and elevates TH mRNA and protein levels (Dermitzaki et al. 2007). Furthermore, in Ucn2 KO mice mRNA levels of adrenomedullary TH and phenylethanolamine N-methyltransferase are significantly decreased compared to WT mice (Riester et al. 2012). However, depending on the dose and duration of treatment, Ucn2 can also act as an inhibitory factor on the catecholaminergic system (Dermitzaki et al. 2007). In addition to an autorcrine/paracrine role in the adrenal medulla, secretion of adrenomedullary-derived Ucn2 into the bloodstream may also have an endocrine function. In this regard, peripheral administration of Ucn2 has a beneficial effect on cardiac function in animal experiments and pilot human studies [reviewed in (Emeto et al. 2011)]. Since Ucn2 has a vasodilatory effect (Kageyama et al. 2003) it may reverse the vasoconstrictive effect of catecholamines released during stress and help the organism restore homeostasis.
In addition to the large IMO-triggered rise in Ucn2, our study demonstrated a significant (about six-fold) rise in adrenomedullary CRH mRNA levels with single and repeated IMO though the significance of these changes may be more modest given the low basal CRH mRNA levels and less profound induction by stress compared to Ucn2. The rise in adrenal CRH gene expression in the adrenal medulla could also influence catecholamine synthesis and release. Activation of CRHR1 in rat chromaffin cells triggered catecholamine secretion and elevated TH mRNA and protein levels (Nanmoku et al. 2005).
Role of glucocorticoids in transcriptional regulation of Ucn2 under stress conditions
HPA axis activation with subsequent elevation of plasma levels of ACTH and corticosterone is a key response of the organism to stress. Our study revealed that treatment of PC12 cells with the synthetic glucocorticoid receptor agonist, DEX, or the endogenous rodent adrenal glucocorticoid, corticosterone, induces a dose-dependent elevation of Ucn2 mRNA. This differs from the negative feedback effects of glucocorticoids on the HPA axis, where DEX lowered Ucn2 mRNA expression levels in cells of the anterior pituitary (Nemoto et al. 2007). The DEX triggered elevation of Ucn2 mRNA levels in PC12 cells is transcriptionally mediated as it was inhibited by actinomycin D. Previously, mouse Ucn2 gene promoter activity was found to be stimulated by DEX and to contain 14 putative glucocorticoid responsive elements (GRE) within the 1.2 kb 5′ flanking region (Chen et al. 2003). Using TESS (Transcription Element Search Software; http://www.cbil.upenn.edu/tess), we found five putative GRE sites within the proximal 1.2 kb 5′ flanking region of the rat Ucn2 gene.
The involvement of glucocorticoids in the IMO-elicited induction of Ucn2 gene expression in the adrenal medulla is supported by results with CRH-KO animals. Mice lacking the CRH gene have low basal corticosterone levels and an impaired glucocorticoid response to various stressors (Muglia et al. 1995, 2000). CRH-KO mice exposed to IMO have an attenuated rise in plasma corticosterone and epinephrine (Kvetnansky et al. 2006), as also shown here for corticosterone. Basal adrenomedullary Ucn2 mRNA levels were similar in both CRH-KO and WT mice. However, the IMO-induced increase in Ucn2 gene expression observed in WT mice was absent in CRH-KO mice, suggesting the involvement of glucocorticoids in induction of Ucn2 gene expression with stress. However, we cannot rule out other CRH requiring mechanisms. In addition to the hypothalamic CRH responsible for the release of ACTH from the pituitary, there are widely dispersed CRH systems in other areas of the brain, such as the brainstem, septum, and amygdala which can mediate physiological responses to stress (Heinrichs and Koob 2004; Orozco-Cabal et al. 2006).
With repeated IMO the rise in glucocorticoids is less sustained after termination of the stress than with the first exposure (Tai et al. 2007), and may explain our findings of smaller rise in Ucn2 mRNA 3 h after 6xIMO compared to 1xIMO (Fig. 1).
In contrast to Ucn2, CRH mRNA levels were significantly decreased in PC12 cells after DEX treatment, although DEX led to a delayed elevation of immunoreactive CRH in a human pheochromocytoma cell line (Venihaki et al. 1998). In rat hypothalamic paraventricular nucleus (PVN) neurons in organotypic culture DEX or corticosterone also markedly reduced CRH mRNA levels (Bali et al. 2008). Nevertheless, we found that CRH mRNA was significantly elevated by IMO. Together this suggests that the IMO-induced elevation in CRH gene expression occurs by a non-glucocorticoid-mediated pathway. This may occur perhaps by way of IMO-triggered activation of CREB in the adrenal medulla (Sabban et al. 2006b), a pathway which mediates stress-triggered induction of CRH gene expression in the PVN (Liu et al. 2010; Aguilera and Liu 2012).
IMO not only elevated CRH and Ucn2 mRNA levels in rat adrenal medulla but also CRHR1 mRNA levels, albeit to a lesser extent of about two-fold. A similar increase was also observed in DEX treated PC12 cells. On the other hand, there was no significant change in CRHR2 gene expression in rat adrenal medulla 3 h after 1x or 6xIMO. However, immediately following 1xIMO, CRHR2 mRNA levels were modestly down-regulated in WT mice, but not in CRH-KO mice. Accordingly, treatment of PC12 cells with DEX decreased CRHR2 gene expression at one of the time points and basal CRHR2 mRNA levels were elevated in unstressed, control CRH-KO mice relative to WT mice. This suggests that IMO triggers a small, transient, glucocorticoid-mediated decrease in CRHR2 gene expression and that furthermore, glucocorticoids may also be important for low basal expression of the CRHR2 gene. In this regard, glucocorticoids can down-regulate CRHR2 in rat heart and aortic smooth muscle (Kageyama et al. 2000).
Overall, our results show that Ucn2 is the most abundant CRH family member mRNA transcript in rat adrenal medulla and that its gene expression increases dramatically in response to stress. The elevated Ucn2, by mechanism which appears to involve stress-triggered rise in glucocorticoids, may be a key mediator of not only adrenomedullary function in response to stress, but may also have an endocrine function.