**2. Endogenous expression of Nrg1 in the anterior pituitary and rat lactosomatotroph GH3 cells**

#### **2.1. Expression and localization of Nrg1 and its receptor in the anterior pituitary of rat and non-human primates**

The Nrg1-ErbB signaling pathway has a critical role in organ development, cell differentiation and tumorigenesis. Neuregulins have previously been described in the nervous system, especially in the cortex, spinal cord and hypothalamus. In the hypothalamus, ARIA (or Nrg1α and β) was expressed in neurons with processes projecting to the posterior pituitary gland but not in those without these projections, suggesting that hypothalamus-derived Neuregulin regulates certain functions of the pituitary (Bernstein et al., 2006; Corfas et al., 1995). Furthermore, Nrg1 receptors were reported to be expressed in hypothalamic astrocytes, where their activation as a result of paracrine Nrg1 stimulation is essential for stimulating secretion of luteinising hormone-releasing hormone (LHRH), intrapituitary gonadotrophin secretion and normal sexual puberty (Bernstein et al., 2006; Prevot et al., 2003).

Neuregulin has also been reported to be expressed in the endocrine organs, including the adrenal gland and the adult pancreas (Harari et al., 1999; Orr-Urtreger et al., 1993). Additionally, thyroid-derived cell lines and corresponding papillary carcinomas also express Nrg1 and ErbB receptors ErbB2 and ErbB4 (Fluge et al., 2000; Mincione et al., 1998). By contrast, Nrg1 expression and localization, as well as its role in the adenohypophyseal structure, have not been fully defined for a long time. Recently, exogenous Nrg1 has been reported to modulate PRL mRNA expression and PRL secretion from the rat lactosomatotroph GH3 cells, where the ErbB3 receptor was shown to correlate with malignant transformation of prolactinomas (Vlotides et al., 2009). Thus, it is essential to elucidate (i) whether the anterior pituitary gland endogenously expresses Nrg1, (ii) whether intrahypophyseal Nrg1ErbB receptor can regulate PRL secretion and (iii) its relevance to the development of prolactinoma.

Neuregulin-1 (Nrg1): An Emerging Regulator of Prolactin (PRL) Secretion 87

presence of both membrane-tethered Nrg1 and soluble Nrg1 may function in an autocrine

Type sample α β S1 S3 S4 ICD-a ICD-b ICD-c I AP + + + + - + + + GH3 - - - - - - - - II AP - - - - - - - - GH3 - - - - - - - - III AP + + + + - + + + GH3 + + + + - + + +

**Table 1.** Nrg1 domain identification in the anterior pituitary (AP) and GH3 cells (GH3) based on domain RT-PCR. α, EGF-like domain α; β, EGF-like domain β; S, stalk domain; ICD, intracellular

**Figure 3. Domain RT-PCR for multiple Nrg1 isoforms in the rat cortex, hypothalamus, anterior pituitary and GH3 cells.** Reverse transcriptase-PCR with several sets of domain specific primers amplified the expression of Nrg1 isoforms in the hypothalamus (HP) and anterior pituitary (AP) and

dehydrogenase (GAPDH) was used to indicate the equal loading of samples. The schematic diagrams right to the RT-PCR results indicate domains that the RT-PCR products contain. α, EGF-like domain α; β, EGF-like domain β; s, stalk domain; CRD, cysteine-rich domain; ICD, intracellular domain; TM,

At the protein level based on Western blot, the anterior pituitary give rise to a group of bands with a wide range of molecular weights as a result of alternative splicing and posttranslational modification such as hyperglycosylation. In the anterior pituitary cell lysates, bands at 140, 110, 95 and 90 kDa, representing the main Nrg1 precursors, were observed, whereas, in the hypothalamus cell lysates, a weak band at 110 kDa was observed. Soluble

GH3 cells (GH3). Rat cortex (Cor) serves as positive control. D-Glyceraldehyde-3-phosphate

transmembrane domain. (Zhao et al., 2011a)

paracrine manner.

domain.

#### *2.1.1. Multiple Nrg1 isoforms are expressed in the anterior pituitary and GH3 cells*

Based on a domain RT-PCR method systematically used by Cote et al. (2005), multiple isoforms of Nrg1 were amplified. In our work, we first amplified a 392-bp band from the rat cortex and anterior pituitary cDNA, corresponding to the spacer domain (SP)-containing Ig-EGFα segment in type I Nrg1 **(Fig 3A)**. When primers spanning the Ig-like domain and the EGFβ domain were used, a band at 401 bp was amplified from the cortex, hypothalamus and anterior pituitary cDNA, representing type I Nrg1β. A 299-bp band was amplified from the cortex and hypothalamus, but not from the anterior pituitary cDNA, which represents the SP free Ig- EGFβ segment exclusively contained in type II Nrg1 **(Fig 3B)**. With primers specific to both the Ig domain and S1, S3 or S4, we found that S1 and S3 are present in type I Nrg1 in rat cortex, hypothalamus and anterior pituitary **(Fig 3C-E)**. When primers against the CRD-EGFα domain was employed, an 833-bp band was weakly amplified from the cortical cDNA and strongly amplified from the anterior pituitary and GH3 cells **(Fig 3G)**. Using primers against the CRD-EGFβ domain, an 842-bp band was amplified in all tested samples **(Fig 3H )**. With primers specific to CRD and S1, S3 or S4, we found that S1 and S3 are present in both forms of type III Nrg1 in rat cortex, hypothalamus, anterior pituitary and GH3 cells **(Fig 3I, J)**, whereas S4 was present in rat cortex and undetectable in the other samples tested **(Fig 1K)**. GAPDH signals were equal in each group, suggesting the equal loading of samples **(Fig 3F)**. To confirm the expression of membrane-anchored Nrg1, transmembrane segments were amplified by using primers specific to the different types of cytoplasmic domains. All samples tested showed two similar bands, in which the upper band represents the TM-cytoplasmic a tail segment and the lower band represents the TMcytoplasmic b tail segment **(Fig 3L)**.

By contrast to previous studies depicting type II Nrg1 (GGF I-III) expression in the pituitary (Goodearl et al., 1993), other studies do not support this idea as a result of the absence or extremely low levels of GGF mRNA in rat pituitary with in situ hybridization (ISH) or RT-PCR (Marchionni et al., 1993). In line with the letter, the rat anterior pituitary expresses both type I Nrg1α β and type III Nrg1α β, whereas the GH3 cells only express type III Nrg1α β (**Tab 1**). This suggests that Nrg1 may have specific functions there. Furthermore, the


presence of both membrane-tethered Nrg1 and soluble Nrg1 may function in an autocrine paracrine manner.

86 Prolactin

development of prolactinoma.

cytoplasmic b tail segment **(Fig 3L)**.

Neuregulin has also been reported to be expressed in the endocrine organs, including the adrenal gland and the adult pancreas (Harari et al., 1999; Orr-Urtreger et al., 1993). Additionally, thyroid-derived cell lines and corresponding papillary carcinomas also express Nrg1 and ErbB receptors ErbB2 and ErbB4 (Fluge et al., 2000; Mincione et al., 1998). By contrast, Nrg1 expression and localization, as well as its role in the adenohypophyseal structure, have not been fully defined for a long time. Recently, exogenous Nrg1 has been reported to modulate PRL mRNA expression and PRL secretion from the rat lactosomatotroph GH3 cells, where the ErbB3 receptor was shown to correlate with malignant transformation of prolactinomas (Vlotides et al., 2009). Thus, it is essential to elucidate (i) whether the anterior pituitary gland endogenously expresses Nrg1, (ii) whether intrahypophyseal Nrg1ErbB receptor can regulate PRL secretion and (iii) its relevance to the

*2.1.1. Multiple Nrg1 isoforms are expressed in the anterior pituitary and GH3 cells* 

Based on a domain RT-PCR method systematically used by Cote et al. (2005), multiple isoforms of Nrg1 were amplified. In our work, we first amplified a 392-bp band from the rat cortex and anterior pituitary cDNA, corresponding to the spacer domain (SP)-containing Ig-EGFα segment in type I Nrg1 **(Fig 3A)**. When primers spanning the Ig-like domain and the EGFβ domain were used, a band at 401 bp was amplified from the cortex, hypothalamus and anterior pituitary cDNA, representing type I Nrg1β. A 299-bp band was amplified from the cortex and hypothalamus, but not from the anterior pituitary cDNA, which represents the SP free Ig- EGFβ segment exclusively contained in type II Nrg1 **(Fig 3B)**. With primers specific to both the Ig domain and S1, S3 or S4, we found that S1 and S3 are present in type I Nrg1 in rat cortex, hypothalamus and anterior pituitary **(Fig 3C-E)**. When primers against the CRD-EGFα domain was employed, an 833-bp band was weakly amplified from the cortical cDNA and strongly amplified from the anterior pituitary and GH3 cells **(Fig 3G)**. Using primers against the CRD-EGFβ domain, an 842-bp band was amplified in all tested samples **(Fig 3H )**. With primers specific to CRD and S1, S3 or S4, we found that S1 and S3 are present in both forms of type III Nrg1 in rat cortex, hypothalamus, anterior pituitary and GH3 cells **(Fig 3I, J)**, whereas S4 was present in rat cortex and undetectable in the other samples tested **(Fig 1K)**. GAPDH signals were equal in each group, suggesting the equal loading of samples **(Fig 3F)**. To confirm the expression of membrane-anchored Nrg1, transmembrane segments were amplified by using primers specific to the different types of cytoplasmic domains. All samples tested showed two similar bands, in which the upper band represents the TM-cytoplasmic a tail segment and the lower band represents the TM-

By contrast to previous studies depicting type II Nrg1 (GGF I-III) expression in the pituitary (Goodearl et al., 1993), other studies do not support this idea as a result of the absence or extremely low levels of GGF mRNA in rat pituitary with in situ hybridization (ISH) or RT-PCR (Marchionni et al., 1993). In line with the letter, the rat anterior pituitary expresses both type I Nrg1α β and type III Nrg1α β, whereas the GH3 cells only express type III Nrg1α β (**Tab 1**). This suggests that Nrg1 may have specific functions there. Furthermore, the **Table 1.** Nrg1 domain identification in the anterior pituitary (AP) and GH3 cells (GH3) based on domain RT-PCR. α, EGF-like domain α; β, EGF-like domain β; S, stalk domain; ICD, intracellular domain.

**Figure 3. Domain RT-PCR for multiple Nrg1 isoforms in the rat cortex, hypothalamus, anterior pituitary and GH3 cells.** Reverse transcriptase-PCR with several sets of domain specific primers amplified the expression of Nrg1 isoforms in the hypothalamus (HP) and anterior pituitary (AP) and GH3 cells (GH3). Rat cortex (Cor) serves as positive control. D-Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used to indicate the equal loading of samples. The schematic diagrams right to the RT-PCR results indicate domains that the RT-PCR products contain. α, EGF-like domain α; β, EGF-like domain β; s, stalk domain; CRD, cysteine-rich domain; ICD, intracellular domain; TM, transmembrane domain. (Zhao et al., 2011a)

At the protein level based on Western blot, the anterior pituitary give rise to a group of bands with a wide range of molecular weights as a result of alternative splicing and posttranslational modification such as hyperglycosylation. In the anterior pituitary cell lysates, bands at 140, 110, 95 and 90 kDa, representing the main Nrg1 precursors, were observed, whereas, in the hypothalamus cell lysates, a weak band at 110 kDa was observed. Soluble Nrg1s at 36 and 30 kDa were detected in the anterior pituitary, whereas only the 36 kDa Nrg1 was detected in the hypothalamus.

Neuregulin-1 (Nrg1): An Emerging Regulator of Prolactin (PRL) Secretion 89

expressed at the highest level during the E1 phase. Both type III Nrg1α and type III Nrg1β were expressed at higher levels during the E1 and E2 phases, when an estrogen surge occurred in response to hypophyseal gonadotrophic hormones. At the protein level, the expression of both 110 kDa and 95 kDa Nrg1s in the anterior pituitary were significantly higher in E1 and E2 phases. No similar expression pattern was observed in the posterior pituitary (Zhao et al., 2011c). In spite of these observations, it is still unclear whether Nrg1 functions in an sex-dependent manner or not in the anterior pituitary, and unfortunately, little is known about the sex-specific expression and function of Nrg1 in the brain (Taylor et

*2.1.2. Localization of Nrg1 and ErbB4 receptor in the anterior pituitary of male Rhesus* 

In male Rhesus monkeys aged 5-7 years, the existence of Nrg1 and ErbB4 was observed, which showed a partial adjacent pattern, suggesting the existence of Nrg1/ErbB4 juxtacrine signaling in the anterior pituitary in non–human primates (See Figure 5) (Zhao et al., 2011c).

**Figure 5. Nrg1 and ErbB4 receptor are expressed in the anteior pituitary of the rhesus monkey.** The anterior pituitary of Rhesus monkey was subjected to immunofluorescence staining for both Nrg1 and

Exogenous Nrg1 was first shown to increase PRL mRNA expression and PRL secretion from GH3 cells by activating the ErbB3 receptor and intracellular AKT. In addition, the ErbB3 receptor has been shown to correlate with the malignant transformation of prolactinomas

Subsequent investigation demonstrated that administration of siRNA against Nrg1 reduced the expression of multiple isoforms, including the 110-, 60-, 36-, 33-, and 30-kDa proteins, indicating that these bands potentially represented alternatively spliced Nrg1 gene

ErbB4 (Green: Nrg1; Red: ErbB4; Blue: DAPI).

(Vlotides et al., 2008, 2009).

**2.2. Expression and Localization of Nrg1 in GH3 cells** 

al., 2012).

*monkeys* 

In the anterior pituitary, Nrg1αβ was co-localized with Nrg1α, further confirming the domain RT-PCR and western blotting results and indicating that Nrg1α is the predominant intrapituitary Nrg1. However, Nrg1αβ was not co-localized with S-100, GH and ACTH, which serve as markers for folliculo-stellate cells, somatotrophs and corticotrophs, respectively. Notably, neighbouring localization of Nrg1 with PRL was observed, suggesting a potential interaction between lactotrophs and Nrg1 positive cells. In addition, Nrg1 was weakly detected in partial PRL positive lactotrophs. Further immunofluoresecence investigation demonstrated Nrg1αβ were co-stained with FSH or LH, both of which are markers for gonadotrophs. Significant co-localization of Nrg1αβ with either FSH or LH was noted in the transition zone between pars tuberalis and pars distalis. However, in the pars distalis, such co-localization was relatively weak. This suggests that gonadotrophs in the pars tuberalis adjacent to the pars distalis are the major source of intrapituitary Nrg1α/β (**Fig 4**) (Zhao et al., 2011a).

**Figure 4. Gonadotrophs are the main source of Nrg1 in the anterior pitutiary.** PL, posterior lobe; IL, intermediate lobe; AL, anterior lobe. A, pars tuberalis; B, pars distalis. (Zhao et al., 2011a)

In addition, RT-PCR demonstrated varying expression patterns of Nrg 1 isoforms at the mRNA level during the estrous cycle. Type I Nrg1α was expressed at a low level during the proestrous (PE) phase and at a constant level in other phases. In contrast, low levels of type I Nrg1β were observed in the metestrous (ME) and diestrous (DE) phases. Type II Nrg1 was expressed at the highest level during the E1 phase. Both type III Nrg1α and type III Nrg1β were expressed at higher levels during the E1 and E2 phases, when an estrogen surge occurred in response to hypophyseal gonadotrophic hormones. At the protein level, the expression of both 110 kDa and 95 kDa Nrg1s in the anterior pituitary were significantly higher in E1 and E2 phases. No similar expression pattern was observed in the posterior pituitary (Zhao et al., 2011c). In spite of these observations, it is still unclear whether Nrg1 functions in an sex-dependent manner or not in the anterior pituitary, and unfortunately, little is known about the sex-specific expression and function of Nrg1 in the brain (Taylor et al., 2012).

88 Prolactin

Nrg1 was detected in the hypothalamus.

intrapituitary Nrg1α/β (**Fig 4**) (Zhao et al., 2011a).

Nrg1s at 36 and 30 kDa were detected in the anterior pituitary, whereas only the 36 kDa

In the anterior pituitary, Nrg1αβ was co-localized with Nrg1α, further confirming the domain RT-PCR and western blotting results and indicating that Nrg1α is the predominant intrapituitary Nrg1. However, Nrg1αβ was not co-localized with S-100, GH and ACTH, which serve as markers for folliculo-stellate cells, somatotrophs and corticotrophs, respectively. Notably, neighbouring localization of Nrg1 with PRL was observed, suggesting a potential interaction between lactotrophs and Nrg1 positive cells. In addition, Nrg1 was weakly detected in partial PRL positive lactotrophs. Further immunofluoresecence investigation demonstrated Nrg1αβ were co-stained with FSH or LH, both of which are markers for gonadotrophs. Significant co-localization of Nrg1αβ with either FSH or LH was noted in the transition zone between pars tuberalis and pars distalis. However, in the pars distalis, such co-localization was relatively weak. This suggests that gonadotrophs in the pars tuberalis adjacent to the pars distalis are the major source of

**Figure 4. Gonadotrophs are the main source of Nrg1 in the anterior pitutiary.** PL, posterior lobe; IL,

In addition, RT-PCR demonstrated varying expression patterns of Nrg 1 isoforms at the mRNA level during the estrous cycle. Type I Nrg1α was expressed at a low level during the proestrous (PE) phase and at a constant level in other phases. In contrast, low levels of type I Nrg1β were observed in the metestrous (ME) and diestrous (DE) phases. Type II Nrg1 was

intermediate lobe; AL, anterior lobe. A, pars tuberalis; B, pars distalis. (Zhao et al., 2011a)

### *2.1.2. Localization of Nrg1 and ErbB4 receptor in the anterior pituitary of male Rhesus monkeys*

In male Rhesus monkeys aged 5-7 years, the existence of Nrg1 and ErbB4 was observed, which showed a partial adjacent pattern, suggesting the existence of Nrg1/ErbB4 juxtacrine signaling in the anterior pituitary in non–human primates (See Figure 5) (Zhao et al., 2011c).

**Figure 5. Nrg1 and ErbB4 receptor are expressed in the anteior pituitary of the rhesus monkey.** The anterior pituitary of Rhesus monkey was subjected to immunofluorescence staining for both Nrg1 and ErbB4 (Green: Nrg1; Red: ErbB4; Blue: DAPI).

#### **2.2. Expression and Localization of Nrg1 in GH3 cells**

Exogenous Nrg1 was first shown to increase PRL mRNA expression and PRL secretion from GH3 cells by activating the ErbB3 receptor and intracellular AKT. In addition, the ErbB3 receptor has been shown to correlate with the malignant transformation of prolactinomas (Vlotides et al., 2008, 2009).

Subsequent investigation demonstrated that administration of siRNA against Nrg1 reduced the expression of multiple isoforms, including the 110-, 60-, 36-, 33-, and 30-kDa proteins, indicating that these bands potentially represented alternatively spliced Nrg1 gene 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 autocrine/paracrine signaling during the development of prolactinoma.

Neuregulin-1 (Nrg1): An Emerging Regulator of Prolactin (PRL) Secretion 91

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

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

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

increase enzymatic cleavage of the PRL precursor.

of PRL, a process that may also occur in the normal anterior pituitary.

receptor dependent manner in cancer cells (Ueno et al., 2008; Yuan et al., 2006).

**cells** 

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

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

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 development.
