Hemokinin-1 Gene Expression Is Upregulated in Microglia Activated by Lipopolysaccharide through NF-κB and p38 MAPK Signaling Pathways

The mammalian tachykinins, substance P (SP) and hemokinin-1 (HK-1), are widely distributed throughout the nervous system and/or peripheral organs, and function as neurotransmitters or chemical modulators by activating their cognate receptor NK1. The TAC1 gene encoding SP is highly expressed in the nervous system, while the TAC4 gene encoding HK-1 is uniformly expressed throughout the body, including a variety of peripheral immune cells. Since TAC4 mRNA is also expressed in microglia, the resident immune cells in the central nervous system, HK-1 may be involved in the inflammatory processes mediated by these cells. In the present study, we found that TAC4, rather than TAC1, was the predominant tachykinin gene expressed in primary cultured microglia. TAC4 mRNA expression was upregulated in the microglia upon their activation by lipopolysaccharide, a well-characterized Toll-like receptor 4 agonist, while TAC1 mRNA expression was downregulated. Furthermore, both nuclear factor-κB and p38 mitogen-activated protein kinase intracellular signaling pathways were required for the upregulation of TAC4 mRNA expression, but not for the downregulation of TAC1 mRNA expression. These findings suggest that HK-1, rather than SP, plays dominant roles in the pathological conditions associated with microglial activation, such as neurodegenerative and neuroinflammatory disorders.


Introduction
Mammalian tachykinins comprise a family of neuropeptides with a common carboxyl terminal amide motif. The representative tachykinin peptide, substance P (SP), is encoded by the TAC1 gene and preferentially activates a G-protein-coupled receptor, NK 1 . The most recently identified tachykinin member, hemokinin-1 (HK-1; encoded by the TAC4 gene), also activates the NK 1 receptor with similar affinity and potency to SP [1,2], although the effects of SP and HK-1 are slightly different [3][4][5][6]. In addition, the TAC4 and TAC1 expression patterns differ considerably. TAC1 is highly expressed in the nervous system, while TAC4 is uniformly expressed throughout the body [7]. TAC4 is expressed in a variety of peripheral immune cells, including lymphocytes, monocytes and macrophages [8][9][10]. Accordingly, HK-1 is involved in immune functions and inflammation [2,5,[11][12][13][14]. However, its role in the nervous system remains almost unknown.
Microglia, the resident immune cells in the central nervous system (CNS), also express the TAC4 gene [10,15], similar to the case for peripheral immune cells. Microglia have significant roles in various neurological diseases, such as neurodegenerative disorders and chronic pain [16,17]. In these situations, microglia become activated to upregulate and release a variety of inflammatory mediators that contribute to the pathophysiology. Therefore, HK-1 expression in microglia may also be involved in the inflammatory processes mediated by these cells in the CNS. In fact, we previously observed that the TAC4 expression level was increased in the dorsal spinal cord in the neuropathic pain state, in which activated microglia underlie the hyperalgesia. The increase in TAC4 expression was suppressed by inhibiting microglial activation [15]. However, it remains unknown how the tachykinin genes are precisely regulated in microglia.
In this study, we examined the expression levels of the TAC1 and TAC4 tachykinin genes in microglia activated by lipopolysaccharide (LPS), a well-characterized Toll-like receptor 4 (TLR4) agonist. TLR4 is a member of the pattern recognition receptors that mediate innate immunity and induces microglial activation in response to pathogen-derived and endogenous molecules. Upon activation by LPS, TAC4 expression was significantly increased in microglia, while TAC1 expression was decreased. Since TLR4induced activation of microglia results in the release of inflammatory substances and mediates various CNS diseases through multiple intracellular mechanisms [18], we further examined the signaling pathways responsible for the regulation of tachykinin expressions and found that the increase in TAC4 expression was mediated by nuclear factor (NF)-kB and p38 mitogen-activated protein kinase (MAPK).

Ethics Statement
All experimental procedures were approved by the Nippon Medical School Animal Care and Use Committee (approval number: 22-054).

Primary microglial culture
Isolated primary microglial cultures were prepared as described previously [19]. Briefly, primary mixed glial cell cultures from whole brains of Sprague-Dawley rats at postnatal day 0-1 were prepared in 75-cm 2 culture flasks and maintained in Eagle's modified essential medium containing 10% fetal bovine serum, 5 mg/ml of bovine insulin and 2% glucose at 37uC in 5% CO 2 . After 2 weeks, the culture flasks were shaken on an orbital shaker at 120 rpm at 37uC for 2 h and the medium was harvested. Subsequently, microglia were recovered as adherent cells to uncoated plastic dishes (Falcon 1001; Becton-Dickinson Japan, Tokyo, Japan). The adherent microglia were collected with a rubber policeman and plated at a concentration of 5610 6 cells per 6-cm plastic dish (Falcon 3002; Becton-Dickinson Japan). The purity of the enriched microglia was more than 97%, as determined by Iba I immunocytochemical staining.

Drug treatment of microglia
At 1 day after plating, the microglia were treated with LPS (0.1 mg/ml), IL-1b (20 mg/ml), ATP (100 mM) or HK-1 (1 mM) for 24 h. To block the intracellular signaling pathways, the microglia were treated with 30 mM SC-514, an NF-kB activation inhibitor, or 10 mM of SB 203580, a p38 MAPK inhibitor, dissolved in dimethyl sulfoxide at 1 h prior to LPS stimulation. Images of the microglial morphology were captured using a high-resolution digital camera equipped with a computer (Olympus, Tokyo, Japan).

Immunocytochemical staining of Iba I
Microglia were seeded onto culture slides and allowed to equilibrate for 24 h. The culture medium was removed and the cells were washed in phosphate-buffered saline (PBS; pH 7.2) and fixed with 4% paraformaldehyde for 15 min at room temperature. After two rinses in PBS, the cells were incubated in the presence of 0.2% Triton X-100 for 10 min, rinsed with PBS twice, blocked with 2% donkey serum for 30 min and incubated with a primary rabbit antibody against Iba I (1:2000 dilution) at 4uC overnight. After three washes in PBS, the cells were incubated with an anti-rabbit IgG antibody conjugated with Alexa Fluor 488 (1:1000 dilution) at room temperature for 1 h. To identify the stained and unstained cells, the slides were mounted in VECTASHIELD Mounting Medium with DAPI, and images were captured using a high-resolution digital camera equipped with a computer (Olympus).

Statistical analysis
Values are presented as means 6 SEM. Differences in the mRNA expression levels were analyzed by an unpaired t-test or one-way repeated-measures ANOVA followed by individual post hoc multiple comparisons (Dunnett's test or Tukey-Kramer multiple comparison test). Values of P,0.05 were considered to indicate statistical significance.
No changes in TAC4 mRNA expression in microglia following IL-1b, ATP or HK-1 treatment In addition to LPS, other molecules such as IL-1b and ATP can also activate microglia [20]. Therefore, we examined the effects of IL-1b and ATP on the TAC4 mRNA expression. However, treatment of the microglia with IL-1b or ATP for 24 h did not significantly increase the TAC4 mRNA expression levels (92.169.3% and 123.5613.1% expressed as percentages of the control, respectively; n = 4-9; Fig. 2). Next, we examined whether the upregulation of TAC4 mRNA expression was induced in autocrine and/or paracrine manners because microglia expressed NK 1 receptors of their own (Fig. 1A). However, HK-1 treatment for 24 h had no effect on the cell number and the expression level of TAC4 mRNA (82.167.3% expressed as a percentage of the control; n = 7-9; Fig. 2).

NF-kB and p38 MAPK inhibitors attenuate the LPSinduced increase in the expression level of TAC4 mRNA
The TLR4 activation induced by LPS is mainly mediated by NF-kB and MAPK pathways [21]. In addition, we previously demonstrated that HK-1 gene expression is increased in the dorsal spinal cord after peripheral nerve injury and blocked by a microglial inhibitor [15]. Among the MAPKs, p38 MAPK is consistently activated by injury in microglia [22]. Therefore, we pretreated microglia with SC-514 and SB 203580 to block the activations of NF-kB and p38 MAPK, respectively. The morphology of nonstimulated microglia was characterized by bipolar or unipolar processes and elongated cell bodies, while microglia activated by LPS exhibited amoeboid cell bodies with retraction of extensions (Fig. 3). In addition, LPS increased the number of microglia. However, the morphological and numerical changes induced by LPS treatment were greatly attenuated in the microglia pretreated with SC-514 or SB 203580 (Fig. 3). The microglial morphology and cell number were unaffected by SC-514 or SB 203580 alone.
Next, we investigated the involvement of the NF-kB and p38 MAPK pathways in the upregulation of TAC4 mRNA expression in the LPS-treated microglia. SC-514 significantly inhibited the upregulation of TAC4 mRNA expression in the LPS-treated microglia (179.0622.8% for LPS alone and 115.968.0% for LPS plus SC-514 expressed as percentages of the control; P,0.05; n = 6-9; Fig. 4A). SB 203580 also significantly attenuated the upregulation of TAC4 mRNA expression (156.067.3% for LPS alone and 130.867.2% for LPS plus SB 203580 expressed as percentages of the control; P,0.05; n = 8-10; Fig. 4B). SC-514 or SB 203580 treatment alone had no effect on the expression levels of TAC4 mRNA (109.3611.4% for SB 203580 and 93.1610.5% for SC-514 expressed as percentages of the control; n = 8; Fig. 4). Pretreatment with dimethyl sulfoxide did not affect the upregulation of TAC4 mRNA expression induced by LPS (94.9623.6% expressed as a percentage of the value for LPS alone; n = 4).
NF-kB and p38 MAPK inhibitors are not involved in the LPS-induced decrease in the expression levels of TAC1, NK 1

and NK 3 mRNAs
We further examined the involvement of NF-kB and p38 MAPK in the downregulation of TAC1, NK 1 and NK 3 mRNA    Fig. 5A), although SC-514 showed a tendency to partially inhibit the downregulation of TAC1 mRNA expression. The decreases in the NK 1 mRNA expression levels were also unaffected by SB 203580 (7.861.6% for LPS alone and 21.364.9% for LPS plus SB 203580 expressed as percentages of the control; n = 6-8; Fig. 5B). On the other hand, SC-514 partially, but significantly, inhibited the downregulation of NK 1 mRNA expression (54.369.6% for LPS plus SC-514 expressed as a percentage of the control; P,0.05; n = 6-8; Fig. 5B).

Discussion
In this study, we found that TAC4, rather than TAC1, was the predominant tachykinin gene expressed in microglia. Furthermore, the TAC4 mRNA expression was upregulated in microglia upon their activation by LPS, while the TAC1 mRNA expression was downregulated. Both the NF-kB and p38 MAPK signaling pathways were required for the upregulation of TAC4 mRNA, but not for the downregulation of TAC1 mRNA.

HK-1 is the predominant tachykinin in activated microglia
In the non-stimulated condition, TAC4 mRNA was expressed in primary cultures of microglia, as previously described for microglial cell lines [10,15]. The expression level was much higher than that of TAC1 mRNA. In addition, the expression of TAC4 mRNA was increased in activated microglia, while TAC1 mRNA expression was decreased. These findings suggest that HK-1 is the predominant endogenous NK 1 receptor ligand expressed in microglia, and that SP and HK-1 are expressed in distinct CNS cells. In fact, SP is preferentially expressed in neurons [23] and its mRNA expression level is much higher than that of TAC4 mRNA expression in the nervous system [7]. On the other hand, TAC4 mRNA is expressed in the resident immunocompetent microglia, similar to the peripheral tissues where a variety of peripheral immune cells, including macrophages and dendritic cells, express TAC4 mRNA [5,[8][9][10]. TAC4 mRNA expression is uniform throughout the whole body, and the intact brain also expresses TAC4 mRNA at comparable amounts to peripheral tissues [7]. In  addition, many reports have intriguingly indicated differences in the effects of SP and HK-1 [3][4][5][6], although both peptides activate NK 1 receptors with comparable high affinity and potency and both NK 2 and NK 3 receptors with lower affinities [1,2]. Therefore, HK-1 may be distinct from SP not only in its expressing cells, but also in its roles in the CNS.
TAC4 mRNA expression was upregulated in microglia activated by the TLR4 agonist LPS, suggesting that HK-1 may mediate the microglial actions on neighboring cells, such as neurons and astrocytes. Although LPS is a component of gramnegative bacterial cell walls, TLR4 was also reported to be activated by various endogenous molecules, including heat shock proteins and high mobility group box 1 protein [18,24]. We previously showed that the expression of TAC4 mRNA was upregulated by peripheral nerve injury in association with microglial activation in the dorsal spinal cord. A microglial activation inhibitor, minocycline, suppressed both the upregulation of HK-1 mRNA and neuropathic pain caused by the injury [15]. Neuropathic pain and microglial activation are also attenuated in TLR4-deficient mice [25]. Intrathecal administration of HK-1 induces nociceptive behavior [6,26]. On the other hand, HK-1 was reported to potentiate the antinociceptive effects of peripheral and supraspinal opioids [27,28]. These findings suggest that an increase in HK-1 expression can actually be induced to mediate the microglial actions in the CNS.

Intracellular signaling pathway for the modulation of HK-1 gene expression
The upregulation of HK-1 gene expression in the microglia activated by LPS required NF-kB and p38 MAPK signaling pathways. NF-kB was shown to be involved in the increase in TAC4 gene transcription by phorbol myristate acetate in a T cell line [29], consistent with the present data showing that NF-kB was prerequisite for LPS-induced HK-1 mRNA upregulation in microglia. In addition, we found that p38 MAPK, which is involved in the transcriptional regulation of a variety of inflammatory cytokines [30], was also required for TAC4 mRNA upregulation. Concomitant inhibition of NF-kB and p38 MAPK may exert synergistic inhibition as in the case of cytokine expression [21]. However, IL-1b, which also induces inflammatory mediators in microglia, did not induce HK-1 gene expression, although both LPS and IL-1b lead to the activation of NF-kB and p38 MAPK [31]. ATP was reported to induce p38 MAPK activation in microglia [20,32]. These findings suggest that LPSinduced TAC4 expression requires other additional signaling molecules that are not recruited by IL-1b. The LPS/TLR4 signaling pathways are composed of myeloid differentiation factor 88 (MyD88)-dependent and -independent pathways [21], while IL-1b functions through the MyD88-dependent pathway [33]. Therefore, the MyD88-independent pathway, which is unique to TLR3 and TLR4, may also be required for the upregulation of TAC4 mRNA expression and downregulation of TAC1 and NK receptors. NF-kB modulates the transcription of target genes, including type I interferons, in a cooperative manner with interferon regulatory factor-3, a transcription factor activated through an MyD88-independent pathway [21,34].
The modulation of HK-1 expression also seems to depend on the cell types. In a pre-B cell line and dendritic cells, TAC4 mRNA expression was reported to be decreased by LPS [5,10]. Although the reason for the discrepancy remains unknown, TLR4 activation reportedly induces different gene expressions via similar intracellular signaling pathways among distinct cell types, such as dendritic cells, microglia and neurons [35]. The expression of TAC4 mRNA in human B lymphocytes is upregulated in chronic lymphocytic leukemia and non-Hodgkin's lymphoma, but downregulated in acute lymphoblastic leukemia [36]. In monocyte and macrophage cell lines, both HK-1 and NK 1 gene expressions are downregulated after co-stimulation with interferon-c, IL-1b and tumor necrosis factor-a [8].

Pathophysiological relevance of HK-1 in microglia
Activated microglia play important roles in neurodegenerative disorders and neuroinflammation, including Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis [16,18]. TLR-induced activation of microglia is responsible for neurotoxic processes in these diseases [18]. On the other hand, microglia also exert neuroprotective effects in situations such as ischemic lesions [37]. The present data indicate that HK-1, rather than SP, may play dominant roles in the pathological and/or restorative situations associated with microglial activation. Therefore, further investigation of the roles of HK-1 in connection with microglial activation may lead to novel insights into the pathophysiology of neurodegenerative and neuroinflammatory disorders.