**3. Autocrine/juxtacrine modulation of prolactin (PRL) secretion via Nrg1/ErbB receptor pathway**

#### **3.1. Modulating role of Nrg1 on PRL secretion in the anterior pituitary**

Previous studies have described the morphological relationship between prolactotrophs and gonadotrophs, which is characterised by conditional gap junctions, and have reported the functional roles of LH on PRL secretion in response to GnRH (Andries et al., 1995; Denef et al., 1983). More recently, Henderson et al. (2008) described GnRH-induced PRL release independent of gonadotrophins.

FSH positive gonadotrophs can form contacts with ErbB3 positive cells and ErbB3 was also shown to be localized on the prolactotroph membrane. These observations, altogether, raised the hypothesis that Nrg1 present in gonadotrophs may function by modulating lactotrophs by interacting with ErbB3 receptor on the membrane. Thus, intrapituitary gonadotrophs and prolactotrophs may partially use gap junctions to form contacts, allowing the binding of gonadotroph-derived membrane-tethered type III Nrg1 to ErbB3 receptors on the prolactotrophic membrane. In addition, both prolactotrophs and gonadotrophs may form contacts through cell adhesion molecules widely distributed in the anterior pituitary, including L1 cell adhesion molecule and neural cell adhesion molecule (Zhao et al., 2010). These molecules may increase the interaction between Nrg1 and ErbB receptors, such as ErbB3 and ErbB4, a process that may activate a series of intracellular signals and also increase enzymatic cleavage of the PRL precursor.

90 Prolactin

development.

**Nrg1/ErbB receptor pathway** 

independent of gonadotrophins.

products, post-translationally modified forms, and/or the shed ectodomains from their initial precursors. Immunofluorescence staining also demonstrated the reduced expression of Nrg1α/β in GH3 cells. Nrg1 was detected, with the ErbB2 receptor partially expressed in some human prolactinoma samples. This suggests the existence of Nrg1/ErbB receptor

In addition, type III Nrg1 (SMDF) is distinct from the other two types of Nrg1 and contains an extra N terminal transmembrane structure. In type III Nrg1, initial proteolysis frees the EGF-like domain from the membrane, leading to juxtacrine signalling characterised by reciprocal intercellular communication (Bao et al., 2003; Hancock et al., 2008). Further cleavage releases a shorter EGF-like domain-containing peptide, which functions in autocrine paracrine interactions. Indeed, high levels of ErbB4 receptor and Nrg1 have also been reported to be expressed in K-ras transformed thyroid Kimol and A6 cells, where Nrg1 signals through the ErbB2/ErbB4 heterodimeric complex in an autocrine manner (Mincione et al., 1998). Although Nrg mRNA was present in both tumor and non-tumor tissue, Nrg precursor isoform immunohistochemically showed nuclear immunostaining in most human papillary carcinomas but not in normal thyroid tissue. Cytoplasmic Nrgα, β1 and β3 were also exclusively detected in papillary carcinomas (Fluge et al., 2000). Significant expression of the ErbB2, ErbB3 and ErbB4 receptors, in addition to Nrg1 isoforms, was also detected in the developing murine fetal pancreas, where they potentially contribute to islet development and regrowth (Kritzik et al., 2000). The strong expression of Nrg1 in lactosomatotroph GH3 tumor cells was in sharp contrast with that observed in the anterior pituitary, where Nrg1 was almost undetectable in the prolactotroph (Zhao et al., 2011b). Thus, overexpression of Nrg1 may play a vitally functional role in prolactinoma

**3. Autocrine/juxtacrine modulation of prolactin (PRL) secretion via** 

Previous studies have described the morphological relationship between prolactotrophs and gonadotrophs, which is characterised by conditional gap junctions, and have reported the functional roles of LH on PRL secretion in response to GnRH (Andries et al., 1995; Denef et al., 1983). More recently, Henderson et al. (2008) described GnRH-induced PRL release

FSH positive gonadotrophs can form contacts with ErbB3 positive cells and ErbB3 was also shown to be localized on the prolactotroph membrane. These observations, altogether, raised the hypothesis that Nrg1 present in gonadotrophs may function by modulating lactotrophs by interacting with ErbB3 receptor on the membrane. Thus, intrapituitary gonadotrophs and prolactotrophs may partially use gap junctions to form contacts, allowing the binding of gonadotroph-derived membrane-tethered type III Nrg1 to ErbB3 receptors on the prolactotrophic membrane. In addition, both prolactotrophs and gonadotrophs may

**3.1. Modulating role of Nrg1 on PRL secretion in the anterior pituitary** 

autocrine/paracrine signaling during the development of prolactinoma.

In one study, type I and type III Nrg1αβ as well as membrane-tethered type III Nrg1 were able to be identified using domain-specific primers-based RT-PCR in the mouse gonadotroph αT3-1 cells. Using Western blot assays with an anti-Nrg1 antibody, a cluster of proteins were observed with molecular weights in the range 30–114 kDa. Proteins at 70, 60 and 45 kDa were also detected in serum-free culture medium conditioned by αT3-1 cells**.** However, commonly recognized soluble Nrg1 with molecular weight ranging from 40–25 kDa were rarely observed, suggesting the precursor is the main form of Nrg1 in this gonadotroph cell line, which may be the base for juxtacrine interaction between Nrg1 and its cognate receptors. Subsequently, PRL and GH secreting GH3 cells were co-cultured with gonadtroph αT3-1 cells pretreated with siRNA against Nrg1. Administration of siRNA against mouse Nrg1 significantly reduced the staining intensities of intracellular Nrg1αβ, as well as their co-localization, as observed with immunofluorescence assays. Nrg1 reduction in αT3-1 cells reduced PRL expression in co-cultured GH3 cells. Co-culturing of GH3 cells with αT3-1 cells treated with siRNA against Nrg1 significantly reduced the secretion of an 18 kDa form of PRL from GH3 cells at 48 h, although it had no significant effect on the secretion of 23-kDa PRL and 22-kDa GH. This result, coupled with the observation that membrane-tethered type III Nrg1 is mainly expressed in the gonadotrophs, suggests the existence of a type III Nrg1-mediated juxtacrine mechanism that affects secretion of a subset of PRL, a process that may also occur in the normal anterior pituitary.

Cleaved full-length PRL has been reported to be a vascular function modulator mainly in the 16-kDa form (Clapp et al., 2006, 2008; Macotela et al., 2006). However, an 18- kDa form was also reported as an intermediate form of the final cleavage product in vitro (Lkhider et al., 2004; Nicoll et al., 1997). We reported that Nrg1 can modulate the release of an 18-kDa cleavage form of PRL, which is typical to GH3 cells. This process may be related to the modulation of Nrg1 on enzymes specific for PRL cleavage, such as cathepsin D and matrix metalloprotease (MMP) family members (Clapp et al., 2006, 2008; Macotela et al., 2006). Indeed, Nrg1 has been shown to promote the expression of MMP-7 and -9 in an ErbB receptor dependent manner in cancer cells (Ueno et al., 2008; Yuan et al., 2006).

#### **3.2. Modulating role of Nrg1 on PRL secretion in rat lactosomatotroph GH3 cells**

In one study, siRNA method was used to investigate the autocrine/paracrine effect of Nrg1 on PRL secretion. siRNA of Nrg1 significantly downregulated the release of a soluble form of 36 kDa Nrg1 into the conditioned culture medium. Western blotting analysis showed significantly reduced secretion of both the 23-kDa and the 18-kDa PRLs into the conditioned culture medium in response to the reduced secretion of 36 kDa Nrg1, and a reduction in

ErbB3 receptor activation was also observed. However, downregulation of Nrg1 has no effects on GH secretion. Thus, Nrg1 may modulate PRL secretion from GH3 cells in an autocrine, paracrine/juxtacrine manner (See Fig 6).

Neuregulin-1 (Nrg1): An Emerging Regulator of Prolactin (PRL) Secretion 93

future investigations. The findings regarding the endogenous expression of Nrg1 and an Nrg1-mediated autocrine/paracrine mechanism in GH3 cells have expanded previous results and reveal Nrg1 as a potential diagnostic serological marker for prolactinoma. In addition, a therapeutic approach involving the direct functional inhibition of Nrg1 might be

Among a series of regulators of PRL, the emerging role of Nrg1 is rather new, but important. Overexpression of Nrg1 and its cognate receptor ErbB2, as well as their co-localization provides the promising therapeutic method to control prolactinoma and hyperprolactinemia. Such a clinical purpose can be achieved by 1) Nrg1 receptor inhibitor, such as Erlotinib, Lapatinib et al.; 2) neutralizing antibody against Nrg1 and 3) their combination. Additionally, Nrg1-mediated autocrine/paracrine mechanism in GH3 cells have expanded previous results and reveal Nrg1 as a potential serological marker not only for prolactinoma diagnosis, but for prognosis evaluation post operation. In addition, a therapeutic approach involving the direct functional inhibition of Nrg1 might be a viable clinical treatment for PRL-secreting pituitary tumors. To avoid the side effects, such as affecting the physiological function of circulating Nrg1 brought by intravenous administration of anti-Nrg1 antibody, the therapeutic hypothesis may be established by intratumoral (i.t.) injection of the therapeutic antibody in patients, whose prolactinomas are highly resistant to chemotherapy or in whom the tumor location can lead to high surgery risk. It has recently been reported that experimental i.t. injections with ErbB2 targeted gold nanoparticles (AuNPs) resulted in high tumor retention with low systemic exposure and

*Center for Neuroscience, Shantou University Medical College, Shantou, Guangdong Province, China* 

Publication of this work was supported by National Natural Science Foundation of China

Andries M, Vijver VV, Tilemans D, Bert C, Denef C. Interaction of alpha-T3-1 cells with lactotropes and somatotropes of normal pituitary in-vitro. Neuroendocrinology 1995;

a viable clinical treatment for PRL-secreting pituitary tumors.

represents an attractive delivery strategy (Chattopadhyay et al., 2012).

*Cedars-Sinai Medical Center, Los Angeles, California 90048, USA* 

**5. Conclusions and perspectives** 

**Author details** 

**Acknowledgement** 

(Project 81171138).

**6. References** 

61: 326–336.

Weijiang Zhao

**Figure 6.** Schematic diagram illustrating the hypothesized model for Neuregulin-1 (Nrg1) on PRL regulation. A, autocrine; P, paracrine; J, juxtacrine.
