Musashi2 binds TGFβR1 regulates mastitis via TGFβ/Smad signaling pathway in dairy cows

doi:10.21203/rs.3.rs-2364816/v1

Download PDF

Research Article

Musashi2 binds TGFβR1 regulates mastitis via TGFβ/Smad signaling pathway in dairy cows

https://doi.org/10.21203/rs.3.rs-2364816/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Objective:

Bacteria-induced mastitis is characterized by mammary pain, swelling of the mammary glands, and decreased milk production and quality in cows. Reducing the negative effects of inflammation is a challenge, and the regulatory mechanism of the inflammatory response in mammary epithelial cells is not yet clear.

Results:

Our results indicated that the RNA-binding protein Musashi2 (Msi2) regulates the inflammatory response and the blood-milk barrier in mastitis. Msi2 is commonly enriched in stem cells and tumor cells. We found that Msi2 expression was upregulated during mastitis. Silencing Msi2 in BMECs and mice increased inflammatory factors and tight junctions. Transcriptional profiling analysis revealed that Msi2 silencing increased transforming growth factor-beta (TGFβ) signaling. RNA-interacting protein immunoprecipitation assays demonstrated that Msi2 could affect the translation of transforming growth factor-beta receptor 1 (TGFβR1), thereby affecting TGFβ signaling. Overall, Msi2 silencing reduced inflammatory reactions and repaired the blood-milk barrier during mastitis

Conclusions:

Our findings indicate that Msi2 plays an important role in mastitis by regulating the TGFβ signaling pathway. A decrease in Msi2 can reduce the negative effects of mastitis by inhibiting the expression of inflammatory factors and increasing tight junction proteins.

Msi2

Mastitis

Inflammation

Tight Junction

TGFβR1

Mastitis is an inflammatory reaction in mammary tissue that is generally associated with lactation and is also known as lactation mastitis. Severe mammary inflammation leads to a decrease or even cessation of milk production and a reduction in milk quality[1, 2]. The accumulation of milk or bacterial infection is the main cause of mastitis[3]. Mastitis presents clinically as pain, redness, and swelling of the mammary gland, and there are mammary abscesses in severe cases[4–6]. Regardless of whether appropriate treatment is given, such as the removal of milk or the use of antibiotics, which are commonly used to treat mastitis, treatment cannot fully alleviate the damage to mammary tissue and restore the loss of milk production and milk quality associated with mastitis[2, 4, 5, 7, 8]. Because mastitis causes serious damage to the health and production of dairy cows, reducing the negative impacts of mastitis is important.

The blood-milk barrier is the most important protective factor of the mammary gland during lactation, and an intact blood-milk barrier is beneficial to the health of the mammary gland and milk quality[9–11]. Tight junctions are important factors in the blood-milk barrier, limiting the passage of pathogens, small molecules, ions, and water through very tight intercellular contacts between epithelial cells, not only regulating the barrier but also acting as signaling platforms to regulate cell growth and differentiation[12–14]. Mammary inflammation can destroy the blood-milk barrier during lactation. Studies have shown that the blood-milk barrier is destroyed during mastitis, which allows pathogenic microorganisms to break through the protective barrier of the mammary gland and exacerbate the inflammatory mammary environment[15, 16]. Studies have shown that mammary inflammation caused by Lipopolysaccharide (LPS) can seriously destroy the blood-milk barrier and exacerbate the inflammatory response of the mammary gland[17–19]. In addition, studies have shown that decreases in small intestinal tight junction proteins and intestinal barrier function lead to intestinal barrier dysfunction, resulting in an inflammatory response that further deteriorates into fibrosis[20]. Similarly, in primary sclerosing cholangitis (PSC), the loss of tight junction proteins is associated with progressive inflammation of the bile ducts, which leads to fibrosis and end-stage liver disease[21]. Epithelial-mesenchymal transition (EMT) is characterized by the loss of cell‒cell contacts and cell polarity, which are hallmarks of tight junction dysfunction[14]. These studies show that disruption of the blood-milk barrier exacerbates mastitis and that exacerbated mastitis also damages the blood-milk barrier. Therefore, repairing the tight junction structure and maintaining the normal physiological function of the barrier may be a potential therapeutic strategy for mastitis.

RNA-binding proteins are involved in posttranslational regulation and can affect the translation or stability of RNA by binding to target genes[22, 23]. The Musashi (Msi) family of RNA-binding proteins is highly expressed in a variety of stem cells, and Musashi2 (Msi2) is a member of this family[22, 24–27]. Msi2 is mainly expressed in hematopoietic stem cells and regulates hematopoietic stem cell fate and hematopoietic function[22, 24]. Several studies have shown that Msi2 can regulate the fate of mesenchymal stem cells[27], promote hair regeneration[26], promote myogenesis[23], and regulate the occurrence and development of intestinal tumors and other tumors[22, 28, 29]. Msi2 interacts at multiple nodes in controlling RNA translation and the transforming growth factor-β (TGF-β) signaling pathway[24, 29, 30]. TGF-β signaling is a strong regulator of EMT, in which tight junction proteins undergo dramatic changes[30]. To date, there has been no report on the role of Msi2 in mammary inflammation.

Previous studies have shown that Msi2 can regulate tight junction proteins to influence cell invasion in non-small cell lung cancer (NSCLC) [30]. Given the critical role of tight junction proteins in the blood-milk barrier, it is a challenge to repair the blood-milk barrier and reduce the mammary inflammatory response in mastitis. Therefore, it is worth exploring whether Msi2 affects tight junction proteins or plays a regulatory role in dairy mastitis in cows.

Animals

Cow mammary tissues were obtained from normal and mastitic cows during the dry milk period in GuangZe Pasture (Changchun, China).

Pregnant ICR female mice (12–14 weeks, SPF grade) were purchased from ChangSheng Biotechnology (Liaoning, China). The mice were first parity pregnant and housed in certified standard laboratory cages. All mice were provided food and water ad libitum before the experiments.

Animal experiments

Lactating mice were randomly divided into four groups after parturition for 3 days: control group (n = 5), LPS (L2630, Sigma‒Aldrich, Saint Louis, MO, USA) treatment group (n = 5) (0.2 mg/mL, 50 µL)[10], LPS + siNC group (n = 5) (10 nmol/40 g), and LPS + siMsi2 group (n = 5) (10 nmol/40 g). On the third day of lactation, the control group was injected with stroke-physiological saline solution, and the three treatment groups were injected with LPS in the fourth pair of mammary glands. After 24 h, the mice were anesthetized with sodium pentobarbital (45 mg/kg). siRNA was injected into mice in the LPS + siNC group and the LPS + siMsi2 group at 24 h and 72 h, respectively. Mammary tissue and blood samples were collected 24 h after the last injection.

The siRNA that was used in animals (Supplementary Table 1) was from chemically modified siRNA synthesized by Ruibo Biotech Co., Ltd. (Guangzhou, China).

Cell culture

Bovine mammary epithelial cells (BMECs) were obtained from cow mammary tissue by the puncture method. The cells were washed with 1× DPBS solution, cut into small pieces, and inoculated into cell culture flasks. After the cells emerged, they were digested with trypsin and used to purify the mammary epithelial cells, which were identified by CK-18 expression. The cells were cultured in DMEM/F12 (01-172-1ACS, BI, Beit Hamek, Israel) containing 10% FBS in a humidified cell incubator with 5% CO₂ at 37°C. After 24 h, 10 µg/mL LPS was added to induce inflammation in mammary epithelial cells, and siRNA or plasmid transfection was performed using Lipofectamine™ 2000 (11668019, Thermo, MA, USA) transfection reagent. The siRNAs for cells (Supplementary Table 2) were purchased from Jima Biotech, Co., Ltd. (Suzhou, China), and the plasmids (Supplementary Table 3) were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China).

RNAseq

TRIzol was used to isolate total RNA from the cells after the treatments. RNA library construction, sequencing, and analysis were performed by Sangon Biological Co., Ltd. (Shanghai, China).

Real-time fluorescent quantitative PCR

Total RNA was isolated from cells using TRIzol reagent (15596018, Invitrogen, MA, USA). First-strand cDNA was synthesized from 600 ng of total RNA using MonScript™ RTIII All-in-One Mix with a dsDNase kit (MR05101M, Monad, Suzhou, China). Real-time reverse transcriptase RT‒PCR was performed using a Bio-Rad CFX96 system. Target gene expression was quantified relative to β-actin.

Western blotting

Modified RIPA buffer (P0013B, Beyotime, Shanghai, China) was used to lyse tissue or cells and collect total proteins. The total protein concentration was then determined with an Enhanced BCA Protein Assay Kit (P0010, Beyotime, Shanghai, China), and an appropriate amount of 4× SDS loading buffer was added according to the protein concentration. The protein was finally denatured by boiling for 10 min. Western blotting was performed with 20 µg of protein according to standard protocols. Rabbit anti-Msi2 (ab76148, abcam), rabbit anti-Occludin (ab216327, abcam), rabbit anti-ZO-1 (ab96587, abcam), rabbit anti-Claudin-3 (83609S, CST), rabbit anti-TGFβR1 (ab31013, abcam), rabbit anti-Smad2/3 (3102s, CST), rabbit anti-p-Smad2/3 (8828, CST), rabbit anti-6×His (K007439P, Solarbio, Beijing, China), rabbit anti-TNFα (11948, CST), rabbit anti-NLRP3 (WL02635, Wanleibio, Shenyang, China), rabbit anti-P-p65 (WL02169, Wanleibio, Shenyang, China), rabbit anti-p65 (8242, CST), rabbit anti-α-Tubulin (ab52866, abcam), rabbit anti-IL-6 (12912, CST), rabbit anti-p38 (8690s, CST), and rabbit anti-P-p38 (4511S, CST) were used.

ELISA

Protein analysis of cow mammary tissue was performed with ELISA kits. According to the manufacturer's instructions, the protein levels of IL-6 (Bovine IL-6 ELISA Kit, SEKB-0365, Solarbio, Beijing, China), IL-1β (Bovine IL-1β ELISA Kit, SEKB-0363, Solarbio, Beijing, China) and TNF-α (Bovine TNF-α ELISA Kit, SEKB-0303, Solarbio, Beijing, China) in dairy cow mammary tissue were measured by the kits.

Myeloperoxidase (MPO) activity assay

Mammary tissue was collected, weighed and processed according to the instructions of the MPO kit (A044-1-1, Jiancheng, Nanjing, China). In brief, the tissue was crushed using a tissue grinder. Then, the supernatant was obtained after centrifugation at 12,000 g/min for 10 min at 4 ℃. Each sample (75 µL) and substrate (75 µL) was added to each well and incubated for 3 to 5 min, followed by the addition of 100 µL of H₂SO₄ (2 M) to terminate the reaction. MPO activity in the samples was measured using a microplate reader at OD 460.

Immunofluorescence and histopathology

Mammary tissue was fixed in 4% paraformaldehyde for 48 h, after which the specimens were embedded in paraffin and cut into 5 µm sections. Immunofluorescence, immunohistochemical staining and HE staining were performed according to the standard protocol. The images were scanned by Pannoramic DESK of 3D HISTECH (P-MIDI P250, Budapest, Hungary), and image processing was performed by Pannoramic Scanner software (Case Viewer: C. V 2.3, C. V 2.0).

Cellular immunofluorescence

Group processing was performed by seeding 10⁵ cells in 12-well plates containing cell slides. Immunofluorescence staining was performed when the cell density was 70%. In brief, after being washed three times with precooled PBS and fixed in 4% paraformaldehyde for 20 min, the cells were permeabilized with 0.5% Triton X-100 for 10 min. The cells were washed 3 times with PBS, the liquid was discarded, goat serum was added dropwise, and the cells were blocked for 1 h at room temperature. The cells were then incubated with rabbit anti-CK18 (ab133263, Abcam, 1:500), rabbit anti-Msi2 (ab76148, Abcam, 1:500), rabbit anti-occludin (ab216327, Abcam, 1:500), rabbit anti-ZO-1 (ab96587, Abcam, 1:500), rabbit anti-claudin-3 (83609S, CST, 1:500) and rabbit anti-TGFβR1 (ab31013, Abcam, 1:500) antibodies overnight at 4°C. The cells were washed three times with PBST the next day. The cells were then incubated with rabbit anti-IgG (4412, CST, 1:1000) for 1 h. The secondary antibodies were incubated and washed three times with PBST. Finally, the cells were stained with DAPI (62248, Thermo, MA, USA) for 5 min and washed three times with PBS. The samples were blotted dry, and the slices were sealed with anti-quenching agent (S36917, Thermo, MA, USA) and photographed by confocal fluorescence microscopy (Olympus FV1000, Tokyo, Japan).

RNA-binding protein immunoprecipitation

RNA-binding protein immunoprecipitation (RIP) assays were performed using the Magna RIP™ Kit (17–701, Magna, Darmstadt, Germany) according to the manufacturer's instructions. Untreated BMECs and BMECs infected with the His-tagged empty plasmid, Msi2 or Msi2RBDmut plasmid (approximately 2.0 × 10⁷ cells) were used. Cells in each group were collected and lysed. We used anti-Msi2 or rabbit normal IgG, and protein A + G magnetic beads that were incubated with RIP cleavage products and placed in RIP wash buffer and rotated overnight at 4 ℃. The next day, the immunoprecipitated protein‒RNA complexes were washed, and the total RNA from the washing of beads was purified. RT‒qPCR analysis was conducted quantitatively.

Statistical analysis

The statistical analysis software was GraphPad Prism. The image data were edited using Adobe Illustrator (AI) software. Comparisons between two groups were performed by unpaired two-tailed Student’s t test, and one-way analysis of variance (ANOVA) was used to compare and analyze three groups of data. P < 0.05 was considered statistically significant.

The RNA binding protein Msi2 is upregulated during mastitis.

We obtained mammary tissue from healthy dairy cows and cows with mastitis and performed histochemical staining and Masson staining. There was no damage to the mammary glands in normal cows (Fig. 1A, B). Compared with those in the normal group, mammary glands in the mastitis group exhibited severe pathological injuries, such as neutrophil infiltration and thickening of the alveolar wall (Fig. 1A). The Masson staining results showed that there were more woven collagen fibers in the mammary tissue of mastitis cows (Fig. 1B). The RT‒qPCR results confirmed that inflammatory cytokines, such as interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-1β (IL-1β), tumor necrosis factor-alpha (TNFα), NOD-like receptor thermal protein domain associated protein 3 (NLRP3), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2), were significantly increased in mastitis mammary tissue (Fig. 1C). The ELISA results showed that blood, milk, and tissue protein levels were consistent with the mRNA levels of mastitis mammary glands, and IL-6, IL-1β, and TNFα protein levels were significantly increased in cows with mastitis (Fig. 1D, E). There has been little research on RNA-binding proteins in mastitis, and we focused on the role of the RNA-binding protein Msi2. Interestingly, in the mammary tissue of cows with mastitis, Msi2 mRNA was increased, and immunohistochemical staining showed that Msi2 protein expression levels were downregulated (Fig. 1F, G).

Msi2 and the inflammatory response are upregulated by LPS in BMECs.

We generated a mastitis model by treating BMECs with 10 µg/mL LPS (Fig. S1). Then, we performed RNA sequencing analysis to compare the gene expression profiles of control and LPS-induced BMECs (Fig. S2). Genes related to the inflammatory response were upregulated, and genes related to the positive regulation of translation and intermediate filament cytoskeleton were downregulated (Fig. S2C, D).

Interestingly, compared with that in the control group, Msi2 was upregulated, and the level of Msi2 expression in BMECs was consistent with the RNA sequencing results and mastitis analysis (Fig. 2A, 1F, G). As expected, inflammatory cytokines were upregulated in LPS-induced BMECs (Fig. 2B, C), suggesting the occurrence of a mastitis-related inflammatory response. Western blotting revealed an increase in the protein level of Msi2 in BMECs with mastitis (Fig. 2D). Immunofluorescence staining revealed increased expression of Msi2 in BMECs with mastitis, which was accompanied by increased protein expression of Msi2 in the nucleus (Fig. 2E). Further analysis showed that the level of phosphorylated p65, an important protein in the NF-κB signaling pathway that is related to inflammation, increased significantly in the LPS group (Fig. 1F). These findings suggest that Msi2 plays a role in the regulation of mastitis.

Msi2 silencing alleviates the LPS-induced inflammatory response in BMECs independent of NF-κB/MAPK signaling.

We successfully silenced Msi2 expression using siRNA (Fig. 3A, B). We found that the mRNA expression of IL-6, IL-1β, TNFα, NLRP3, INOS, and COX-2 in BMECs decreased after Msi2 was silenced (Fig. 3C, D). Similarly, the protein expression of MSI2 in the nucleus was reduced significantly (Fig. 3E). However, compared with the reference protein, the levels of phosphorylated p38 and p65 did not change significantly (Fig. 3F, G). Taken together, these results suggest that Msi2 is required for mastitis, but it probably does not require the NF-κB/MAPK signaling pathway.

We used Gene set enrichment analysis (GSEA) to mine the RNA-seq data, and the results showed that Msi2 knockdown increased the enrichment scores for the NF-κB and MAPK signaling pathway modules (Fig. 3H, I). However, western blot analysis of P-p65/p65 and P-p38/p38 showed no significant differences (Fig. 3F, G). Due to the increased degree of fibrosis in the tissues of cows with mastitis (Fig. 1B), we focused on the TGFβ signaling pathway, which is closely related to the increase in fibrosis in dairy cows with mammary gland inflammation. Significantly upregulated genes, as determined by GSEA, are visualized by a heatmap (Fig. 3K).

Silencing Msi2 restores the decrease in tight junctions in mastitis.

We next examined whether tight junction expression changed during mastitis. We induced mastitis in BMECs with LPS and found that ZO-1, Occludin, Symplekin, and Claudin-3 expression levels decreased, as indicated by RT‒qPCR and Western blotting (Fig. 4A, B). Immunofluorescence staining revealed decreased expression levels of ZO-1, Occludin, and Claudin-3 in BMECs with mastitis (Fig. 4C - E). Moreover, we examined the expression of ZO-1, Occludin, and Claudin-3 in the tissues of cows with mastitis by immunohistochemistry and found mammary epithelial cell aggregation and decreased protein levels in these samples (Fig. S3). In contrast, these changes in tight junctions were reversed after Msi2 was silenced. In other words, the Occludin, Symplekin, and Claudin-3 expression levels increased in BMECs treated with siMsi2, as indicated by RT‒qPCR and Western blotting, but ZO-1 was not affected (Fig. 4F, G). Immunofluorescence staining revealed increased expression levels of ZO-1, Occludin, and Claudin-3 in BMECs treated with siMsi2 (Fig. 4H - J). These data suggest that the RNA binding protein Msi2 plays an important role in the blood-milk barrier of the mammary gland.

Msi2 affects inflammatory factors and tight junctions in BMECs via TGFβ-Smad2/3 signaling.

We further analyzed the changes in the TGFβ signaling pathway in BMECs. TGFβR1 and Smad3 are important receptors and downstream factors of the TGFβ signaling pathway. The expression levels of the TGFβR1 and Smad3 were further confirmed in BMECs with mastitis by RT‒PCR (Fig. 5A). Consistent with the changes in the mRNA and protein expression of TGFβR1 and smad3, these levels were increased by LPS and decreased by Msi2 silencing (Fig. 5A, B). The immunofluorescence staining results showed that TGFβR1 was highly expressed in the nucleus after LPS induction but was decreased after Msi2 silencing (Fig. 5C).

Next, we used the pcDNA3.1 vector to construct the Msi2 overexpression plasmid, and the specific TGFβR1 inhibitor SB431542 was used to treat BMECs. The mRNA (Fig. S4A - C) and protein expression levels of Msi2 were increased by the Msi2 plasmid with or without the TGFβR1 inhibitor (Fig. 5D). In BMECs, compared with those in the control group, the mRNA levels of inflammatory factors (IL-6, IL-1β, TNFα, NLRP3, COX-2, INOS) were increased significantly by MSI2 overexpression (Fig. 5F, G). The addition of SB431542 decreased inflammatory factors, but the change was not significant (Fig. 5F, G). Compared with that in the group that was transfected with the pcDNA-Msi2 plasmid alone, the increase in inflammatory factors induced by MSI2 overexpression was inhibited when MSI2 was overexpressed and SB431542 was added to BMECs (Fig. 5F, G). The protein levels of Occludin and Claudin-3 showed similar changes; these factors were suppressed after msi2 overexpression and rebounded after TGFβRI inhibition (Fig. 5H). Because TGFβR1 is considered to be one of the major drivers of tight junctions, these results suggest that Msi2 may regulate the blood-milk barrier and mastitis via the TGFβ-smad2/3 signaling pathway.

Msi2 inhibits TGFβR1 translation and mRNA stability in BMECs.

Previous results demonstrated that Msi2-RNA binding required the three phenylalanine residues in Msi2. Using T-coffee alignment, we found that Msi2 was highly conserved in mammals (Fig. 6A). The three phenylalanine residues were mutated to leucine (F60/62/65L) to synthesize a mutant with Msi2-RNA binding deficiency (Fig. 6A)[31, 32]. Then, we examined the function of Msi2 and the Msi2 mutant by RT‒qPCR and Western blotting. Msi2 overexpression induced the expression of IL-6, IL-1β, and TNFα, but Msi2 mutant overexpression did not induce IL-6, IL-1β, and TNFα expression (Fig. 6B). Moreover, Msi2 overexpression reduced the protein levels of TGFβR1, TNFα, Occludin, and Claudin-3 in BMECs (Fig. 6C, D). In contrast, Msi2 mutant overexpression abolished the inhibitory effect of Msi2 (Fig. 6C, D). These results show that the mRNA binding activity of Msi2 is required for mastitis.

Msi2 has been thought to regulate translation by binding the 3’-UTR of the target mRNA[32, 33]. The MBEs in the 3’ UTR, which are required for Msi2 binding, were found in TGFβR1 mRNA (Fig. 6E). We then performed an RNA immunoprecipitation (RIP) assay using BMECs transfected with plasmids expressing His-Msi2 or His-Msi2RBDmut. As expected, TGFβR1 transcripts were significantly enriched by His immunoprecipitation when His-Msi2 was expressed. In contrast, TGFβR1 transcripts were not enriched when the His-Msi2 mutant was expressed (Fig. 6F). These results suggest that Msi2 binds to the mRNA of TGFβR1. Consistently, RIP with an anti-Msi2 antibody specifically enriched TGFβR1 transcripts relative to that of the immunoglobulin-G (IgG) control (Fig. 6F). Msi2 overexpression in BMECs was performed and changed the mRNA stability of TGFβR1 (Fig. 6G). These results indicate that the binding of Msi2 to TGFβR1 transcripts positively regulates the translation of TGFβR1. The regulation of TGFβ signaling by Msi2 is essential for inflammatory factors and tight junctions in mastitis.

Msi2 regulates mastitis by affecting tight junction proteins in vivo.

The siRNA was modified for use in vivo and was injected into mouse mammary glands. Figure 7A shows a schematic diagram of the mouse treatment cycle. Briefly, LPS was injected into the fourth pair of mammary glands of ICR mice after the first delivery and 3 days of lactation, and siRNA was injected every 48 h twice after 24 h, then the samples were collected. The blood biochemical results showed that LPS successfully induced an inflammatory response in mammary tissue (Fig. S5A, B). The histomorphological results showed that the mammary tissue of the LPS and siNC + LPS groups was red, vascularly congested, and swollen compared to that of the control group, indicating inflammation in the mammary tissue. Significantly, the pathological phenotype was attenuated by Msi2 silencing in mice (Fig. 7B). Moreover, histochemical staining showed that gland acini in the LPS and siNC + LPS groups were significantly atrophied, neutrophil infiltration and thickening of the alveolar wall were observed, mammary tissue was damaged compared with that in the control group, and siMsi2 treatment significantly attenuated this phenotype (Fig. 7B).

The mRNA expression of Msi2 in mouse mammary tissue increased after LPS treatment, and silencing siMsi2 significantly reduced the LPS-induced increase in Msi2 (Fig. 7C). Consistent with the pathological findings, the MPO activity results indicated that silencing MSI2 reduced LPS-induced inflammation (Fig. 7E). The same was true for the expression of inflammatory factors (IL-6, IL-1β, TNFα, and NLRP3) and tight junction proteins (Occludin, Claudin-3) as determined by RT‒qPCR (Fig. 7F). The protein levels of inflammatory factors increased with increasing LPS treatment and were decreased by silencing Msi2 (Fig. 7F). These results indicate that the RNA binding protein Msi2 plays an important role in mastitis.

The acute onset and severe course of mastitis severely affect the health and milk production of cows[2, 8, 34, 35]. Exploring the molecular mechanisms of mastitis is important for finding ways to protect mammary health and mitigate production losses. Here, we reveal the role of Msi2 in LPS-induced mastitis. Msi2 is highly expressed in mammary tissue and epithelial cells in inflamed mammary glands. After silencing Msi2 in mammary epithelial cells and mice, tight junction proteins in mammary tissues were repaired, and mammary inflammation was reduced. Msi2 specifically binds to the 3'UTR of TGFβ, the receptor of TGFβ, affecting its mRNA stability and promoting its translation, thereby activating TGFβ-smad2/3 signal transduction. In conclusion, we found that during mastitis, elevated Msi2 expression is associated with mammary blood-milk barrier disruption and inflammation, and reducing Msi2 expression can repair the blood-milk barrier and alleviate mastitis.

The health of the mammary gland and milk depends on the blood-milk barrier, an important biological barrier in the mammary gland, and tight junction proteins are important components[13, 36]. ZO-1 is a scaffolding protein, Occludin controls the permeability of relatively large molecules, and the Claudin family controls paracellular ion selectivity; collectively, these factors maintain the blood-milk barrier by playing their respective roles during the inflammatory response in the mammary gland[37]. Increased expression of Msi2 in mammary tissue and BMECs predicts the disruption of this balance and an increase in mammary inflammation. In mammary tissue, a prolonged inflammatory response disrupts the homeostatic balance of the tissue microenvironment until tissue fibrosis is induced[38–40]. NLRP3 recognizes relevant mastitis risk molecules and induces the production of the inflammatory factors IL-6, IL-1β, and TNF-α[41]. BMECs with elevated Msi2 levels indicate the activation of the cell's immune capacity. The production of the inducible enzymes iNOS and COX-2 following the stimulation of mammary tissue or cells further leads to severe responses and tissue damage[42]. This evidence corroborates the occurrence of mammary inflammation and fibrosis in mammary tissue caused by severe mammary inflammation. Our results suggest that the RNA binding protein Msi2 is positively associated with mastitis and that inhibiting Msi2 significantly attenuates the increases in IL-6, IL-1β, TNF-α, iNOS, COX-2, NLRP3, and MPO in breast tissue and effectively controls the formation of the inflammatory microenvironment. Silencing Msi2 significantly attenuates LPS-mediated inhibition of the tight junction proteins Occludin, Claudin-3, and ZO-1 and disruption of the blood-milk barrier. The current treatment of mastitis is also focused on suppressing the inflammatory response, which is consistent with the aim of our study[2, 35].

RNA-binding proteins play important roles in inflammation via posttranscriptional regulation[43]. The Msi family is an evolutionarily conserved RNA-binding protein family, especially in mammals[23]. Msi2 has a conserved structure with two tandem RNA recognition motifs and a carboxy-terminal poly-A-binding protein binding domain[22, 24]. This structure allows Msi2 to bind to the r(G/A)U ₁₋₃ AGU sequence (Msi binding element, MBE) in the 3'-untranslated region (3'-UTR) of the target mRNA to regulate poly-A binding protein from entering the extended portion of the translation initiation complex [24, 31, 32]. Previous studies of Msi2 have focused on its functions in tumors, such as maintaining stem cell self-renewal[23, 32, 44]. Recently, studies have shown that Msi2 controls HSC fate through TGF-β signaling[24] and positive feedback regulation in glioblastoma[29]. In non-small cell lung cancer (NSCLC), Msi2 regulates EMT through the claudin family, which in turn affects NSCLC cell invasion[30]. In mastitis, TGF-β/Smad is the classical fibrosis signaling pathway and regulates the inflammatory response through a nonclassical pathway[45]. The expression level of TGF-β1 is significantly increased in inflamed tissues[45], Smad 3 is regulated by TGF-β1, and TGF-βR1 is an important receptor for TGF-β1[45]. TGF-β1 binds to TGF-βR1 and induces the phosphorylation of Smad 3 to form the Smad complex, which translocates to the nucleus to activate and promote EMT, disrupting cell polarity and exacerbating the tight junction protein imbalance[45, 46]. Our findings show that Msi2, TGF-β/Smad signaling, and the classical inflammatory signal NF-κB/MAPK are elevated during mastitis. Silencing Msi2 decreased the inflammatory response but did not show significant changes in NF-κB/MAPK signaling, while TGF-βR1 and Smad 3 changed strongly. Further experiments revealed that Msi2 specifically bound to the 3' UTR of TGFβR1, and the regulation of tight junction proteins and inflammatory responses by Msi2-targeted TGF-βR1 was mediated by the TGF-β/Smad signaling pathway.

In this study, we report the regulatory role of Msi2 in mammary inflammation for the first time, and this factor plays a crucial role in the inflammatory response the homeostasis of the blood-milk barrier. Our study revealed that Msi2 specifically binds to the 3' UTR of TGFβR1 to regulate the TGF-β/Smad signaling pathway, as well as the integrity of the blood-milk barrier, and controls the inflammatory response to mastitis. There are some limitations of this study. We used siRNA to silence Msi2 in the mammary glands of mice. We did not construct knockout mice, which would limit our observation of the mastitis phenotype in vivo without Msi2 in the mammary gland. However, our in vivo and in vitro experiments showed that Msi2 was silenced in mammary epithelial cells, which resulted in a mastitis phenotype. Alternatively, direct genetic evidence is needed for a link between Msi2 and TGF-β signaling.

This study demonstrated that the RNA binding protein Msi2 plays a significant role in mastitis. Msi2 regulates activation of the TGF-β/Smad signaling pathway by binding TGFβR1, maintaining the blood-milk barrier, and controlling the inflammatory microenvironment. This work defined the role of Msi2 in regulating mastitis and identified a new target for tight junctions in mastitis (Fig. 8).

Msi2

Musashi2

BMECs

Bovine mammary epithelial cells

TGFβ

Transforming growth factor-beta

TGFβR1

Transforming growth factor-beta receptor 1

LPS

Lipopolysaccharide

PSC

Primary sclerosing cholangitis

EMT

Epithelial-mesenchymal transition

siRNA

Small interfering RNA

MPO

Myeloperoxidase

IgG

Immunoglobulin G

Histopathology

RIP

RNA-binding protein immunoprecipitation

IL-6

Interleukin-6

IL-8

interleukin-8

IL-1β

Interleukin-1β

TNFα

Tumor necrosis factor-alpha

NLRP3

NOD-like receptor thermal protein domain associated protein 3,

iNOS

Inducible nitric oxide synthase

COX-2

Cyclooxygenase-2

GSEA

Gene set enrichment analysis

MBE

Msi binding element

NSCLC

Non-small cell lung cancer

Ethics approval and consent to participate

All animal care and experimental procedures were performed according to the requirements of the Experimental Animal Ethics and Welfare Committee of Jilin University (approval no.: SY202111023).

Consent for publication

Not applicable.

Availability of data and material

The datasets used and analysed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no competing interests.

Funding

Supported by the earmarked fund for Chinese Agricultural Science Research System (CARS-37) and National Natural Science Foundation of China (U20A2053). The funding was provided for the design of the study, collection, analysis of data and in writing the manuscript.

Authors' contributions

CL and BY were major contributors in writing the manuscript and in performing the experiments. HC performed the experiments of western blotting. YM performed the experiments of RT-qPCR. YZ analyzed the data of RNAseq. YH, SC,ZJ and XJ analyzed the data and contributed reagents. CL and BY revised the article. Hao Jiang and Bao Yuan supervised the study. All authors agree with the content of the manuscript for publication.

Acknowledgements

We thank all those who helped in the experiments, especially Prof. ShouPeng Fu who provided the mammary tissue of the cow.

Li C, Lian L, Kunlin C, et al. UFL1 Alleviates Lipopolysaccharide-Induced Cell Damage and Inflammation via Regulation of the TLR4/NF-κB Pathway in Bovine Mammary Epithelial Cells [J]. Oxidative Medicine and Cellular Longevity, 2019, 2019: 6505373. https://doi.org/10.1155/2019/6505373.
Ruegg PL. A 100-Year Review: Mastitis detection, management, and prevention [J]. J Dairy Sci. 2017;100(12):10381–97. https://doi.org/10.3168/jds.2017-13023.
Foxman Betsy D'Arcy, Hannah G, Brenda, et al. Lactation Mastitis: Occurrence and Medical Management among 946 Breastfeeding Women in the United States [J]. Am J Epidemiol. 2002;155(2):103–14. https://doi.org/10.1093/aje/155.2.103.
Spencer JP. Management of mastitis in breastfeeding women [J]. Am Fam Physician. 2008;78(6):727–31.
Vogel A, Hutchison BL, Mitchell EA. Mastitis in the first year postpartum [J]. Birth. 1999;26(4):218–25. https://doi.org/10.1046/j.1523-536x.1999.00218.x.
Cao Yu, Zhang Y, Lijun M, et al. Niacin stimulates EPH4EV mammary epithelial cell proliferation and mammary gland development in pubertal mice through activation of AKT/mTOR and ERK1/2 signaling pathways [J]. Cell Tissue Res. 2021;384(2):313–24. https://doi.org/10.1007/s00441-020-03355-x.
Olsen CG, Gordon RE. Jr. Breast disorders in nursing mothers [J]. Am Fam Physician. 1990;41(5):1509–16.
Arroyo R, Martín V, Maldonado A, et al. Treatment of infectious mastitis during lactation: antibiotics versus oral administration of Lactobacilli isolated from breast milk [J]. Clin Infect Dis. 2010;50(12):1551–8. https://doi.org/10.1086/652763.
Kobayashi K, Kumura H. Distinct behavior of claudin-3 and – 4 around lactation period in mammary alveolus in mice [J]. Histochem Cell Biol. 2011;136(5):587–94. https://doi.org/10.1007/s00418-011-0863-6.
Guo W, Li W, Su Y, et al. GPR109A alleviate mastitis and enhances the blood milk barrier by activating AMPK/Nrf2 and autophagy [J]. Int J Biol Sci. 2021;17(15):4271–84. https://doi.org/10.7150/ijbs.62380.
Guo W, Liu B, Yin Y, et al. Licochalcone A Protects the Blood-Milk Barrier Integrity and Relieves the Inflammatory Response in LPS-Induced Mastitis [J]. Front Immunol. 2019;10:287. https://doi.org/10.3389/fimmu.2019.00287.
Zihni Ceniz M, Clare M. Karl, et al. Tight junctions: from simple barriers to multifunctional molecular gates [J]. Nat Rev Mol Cell Biol. 2016;17(9):564–80. https://doi.org/10.1038/nrm.2016.80.
Gonschior Hannes S, Christopher, Van der Veen Rozemarijn Eva. et al. Nanoscale segregation of channel and barrier claudins enables paracellular ion flux [J]. Nat Commun. 2022;13(1):4985. https://doi.org/10.1038/s41467-022-32533-4.
Lee DBN, Edmund H, Ward Harry J. Tight junction biology and kidney dysfunction [J]. Am J Physiology-Renal Physiol. 2006;290(1):F20–34. https://doi.org/10.1152/ajprenal.00052.2005.
Jiang A, Zhang Y, Zhang X, et al. Morin alleviates LPS-induced mastitis by inhibiting the PI3K/AKT, MAPK, NF-κB and NLRP3 signaling pathway and protecting the integrity of blood-milk barrier [J]. Int Immunopharmacol. 2020;78:105972. https://doi.org/10.1016/j.intimp.2019.105972.
Stelwagen K, Singh K. The role of tight junctions in mammary gland function [J]. J Mammary Gland Biol Neoplasia. 2014;19(1):131–8. https://doi.org/10.1007/s10911-013-9309-1.
Wall SK, Hernández-Castellano LE, Ahmadpour A, et al. Differential glucocorticoid-induced closure of the blood-milk barrier during lipopolysaccharide- and lipoteichoic acid-induced mastitis in dairy cows [J]. J Dairy Sci. 2016;99(9):7544–53. https://doi.org/10.3168/jds.2016-11093.
Wang JJ, Wei ZK, Zhang X, et al. Butyrate protects against disruption of the blood-milk barrier and moderates inflammatory responses in a model of mastitis induced by lipopolysaccharide [J]. Br J Pharmacol. 2017;174(21):3811–22. https://doi.org/10.1111/bph.13976.
Tsugami Yusaku W, Haruka K, Manabu, et al. Adverse effects of LPS on membrane proteins in lactating bovine mammary epithelial cells [J]. Cell Tissue Res. 2021;384(2):435–48. https://doi.org/10.1007/s00441-020-03344-0.
Liu Yang, Cavallaro Paul M, Byeong-Moo K, et al. A role for intestinal alkaline phosphatase in preventing liver fibrosi s [J]. Theranostics11 (1): 14–26. https://doi.org/10.7150/thno.48468.
Reich M, Spomer L, Klindt C, et al. Downregulation of TGR5 (GPBAR1) in biliary epithelial cells contributes to the pathogenesis of sclerosing cholangitis [J]. J Hepatol. 2021;75(3):634–46. https://doi.org/10.1016/j.jhep.2021.03.029.
Fox RG, Park FD, Koechlein CS, et al. Musashi signaling in stem cells and cancer [J]. Annu Rev Cell Dev Biol. 2015;31:249–67. https://doi.org/10.1146/annurev-cellbio-100814-125446.
Yang W, Yang L, Wang J, et al. Msi2-mediated MiR7a-1 processing repression promotes myogenesis [J]. J Cachexia Sarcopenia Muscle. 2022;13(1):728–42. https://doi.org/10.1002/jcsm.12882.
Park SM, Deering RP, Lu Y, et al. Musashi-2 controls cell fate, lineage bias, and TGF-β signaling in HSCs [J]. J Exp Med. 2014;211(1):71–87. https://doi.org/10.1084/jem.20130736.
de Andrés-Aguayo L, Varas F, Kallin EM, et al. Musashi 2 is a regulator of the HSC compartment identified by a retroviral insertion screen and knockout mice [J]. Blood. 2011;118(3):554–64. https://doi.org/10.1182/blood-2010-12-322081.
Ma X, Tian Y, Song Y, et al. Msi2 Maintains Quiescent State of Hair Follicle Stem Cells by Directly Repressing the Hh Signaling Pathway [J]. J Invest Dermatol. 2017;137(5):1015–24. https://doi.org/10.1016/j.jid.2017.01.012.
Hope KJ, Sauvageau G. Roles for MSI2 and PROX1 in hematopoietic stem cell activity [J]. Curr Opin Hematol. 2011;18(4):203–7. https://doi.org/10.1097/MOH.0b013e328347888a.
Dong W, Liu X, Yang C, et al. Glioma glycolipid metabolism: MSI2-SNORD12B-FIP1L1-ZBTB4 feedback loop as a potential treatment target [J]. Clin Transl Med. 2021;11(5):e411. https://doi.org/10.1002/ctm2.411.
Jiang X, Tan J, Wen Y, et al. MSI2-TGF-β/TGF-β R1/SMAD3 positive feedback regulation in glioblastoma [J]. Cancer Chemother Pharmacol. 2019;84(2):415–25. https://doi.org/10.1007/s00280-019-03892-5.
Kudinov Alexander E, Alexander D, Nikonova Anna S, et al. Musashi-2 (MSI2) supports TGF-β signaling and inhibits claudins to promote non-small cell lung cancer (NSCLC) metastasis [J]. Proceedings of the National Academy of Sciences, 2016, 113 (25): 6955–6960. https://doi.org/doi:10.1073/pnas.1513616113.
Hattori A, Tsunoda M, Konuma T, et al. Cancer progression by reprogrammed BCAA metabolism in myeloid leukaemia [J]. Nature. 2017;545(7655):500–4. https://doi.org/10.1038/nature22314.
Suo J, Zou S, Wang J, et al. The RNA-binding protein Musashi2 governs osteoblast-adipocyte lineage commitment by suppressing PPARγ signaling [J]. Bone Res. 2022;10(1):31. https://doi.org/10.1038/s41413-022-00202-3.
Fox Raymond G, Park Frederick D, Koechlein Claire S, et al. Musashi Signaling in Stem Cells and Cancer [J]. Annu Rev Cell Dev Biol. 2015;31(1):249–67. https://doi.org/10.1146/annurev-cellbio-100814-125446.
Kan X, Hu G, Huang B, et al. Pedunculoside protects against LPS-induced mastitis in mice by inhibiting inflammation and maintaining the integrity of blood-milk barrier [J]. Aging. 2021;13(15):19460–74. https://doi.org/10.18632/aging.203357.
Jahanfar S, Ng CJ, Teng CL Antibiotics for mastitis in breastfeeding women [J]. Cochrane Database Syst Rev, 2013, (2): Cd005458. https://doi.org/10.1002/14651858.CD005458.pub3.
Zihni C, Mills C, Matter K, et al. Tight junctions: from simple barriers to multifunctional molecular gates [J]. Nat Rev Mol Cell Biol. 2016;17(9):564–80. https://doi.org/10.1038/nrm.2016.80.
Zou Jinjing L, Ye Yu, Jimian, et al. Idiopathic pulmonary fibrosis is associated with tight junction protein alterations [J]. Biochimica et Biophysica Acta (BBA) - Biomembranes, 2020, 1862 (5): 183205. https://doi.org/https://doi.org/10.1016/j.bbamem.2020.183205.
Sfanos KS, Yegnasubramanian S, Nelson WG, et al. The inflammatory microenvironment and microbiome in prostate cancer development [J]. Nat Rev Urol. 2018;15(1):11–24. https://doi.org/10.1038/nrurol.2017.167.
Suman S, Sharma PK, Rai G, et al. Current perspectives of molecular pathways involved in chronic inflammation-mediated breast cancer [J]. Biochem Biophys Res Commun. 2016;472(3):401–9. https://doi.org/10.1016/j.bbrc.2015.10.133.
Yan L, Xu F, Dai CL. Relationship between epithelial-to-mesenchymal transition and the inflammatory microenvironment of hepatocellular carcinoma [J]. J Exp Clin Cancer Res. 2018;37(1):203. https://doi.org/10.1186/s13046-018-0887-z.
Seoane PI, Lee B, Hoyle C, et al. The NLRP3-inflammasome as a sensor of organelle dysfunction [J]. J Cell Biol, 2020, 219 (12). https://doi.org/10.1083/jcb.202006194.
Walker CCF, Brester JL, Sordillo LM. Flunixin Meglumine Reduces Milk Isoprostane Concentrations in Holstein Dairy Cattle Suffering from Acute Coliform Mastitis [J]. Antioxid (Basel), 2021, 10 (6). https://doi.org/10.3390/antiox10060834.
Zhongfei M, Yumei Z. Wang Yuyang, et al. RNA-binding protein hnRNP UL1 binds κB sites to attenuate NF-κB-mediated inflammation [J]. J Autoimmun. 2022;129:102828. https://doi.org/https://doi.org/10.1016/j.jaut.2022.102828.
Jiang Xingjun T, Jun W, Yin, et al. MSI2-TGF-β/TGF-β R1/SMAD3 positive feedback regulation in glioblastoma [J]. Cancer Chemother Pharmacol. 2019;84(2):415–25. https://doi.org/10.1007/s00280-019-03892-5.
Kubiczkova L, Sedlarikova L, Hajek R, et al. TGF-beta - an excellent servant but a bad master [J]. J Transl Med. 2012;10:183. https://doi.org/10.1186/1479-5876-10-183.
Geng XQ, Ma A, He JZ, et al. Ganoderic acid hinders renal fibrosis via suppressing the TGF-β/Smad and MAPK signaling pathways [J]. Acta Pharmacol Sin. 2020;41(5):670–7. https://doi.org/10.1038/s41401-019-0324-7.

FIGURES1.png
Fig S1. Construction of the mastitis model in BMECs. (A) Immunofluorescence analysis of CK-18 (green) and DAPI (blue) in BMECs after LPS treatment. Scale bar=50 μm. (B) Real-time cell analysis (RTCA) was used to create the cell proliferation curve in the iCELLigence system. The data represent the mean ± SD (*p<0.05,^$p<0.01, compared with the respective controls), unpaired Student’s t test. (C - D) Representative flow cytometry images of cell apoptosis levels in BMECs stimulated with different concentrations of LPS. The data represent the mean ± SD (**p<0.01, compared with the control) unpaired Student’s t test. (E) qPCR results showing IL-6, IL-1β, and TNFα expression in BMECs after treatment with different concentrations of LPS. The data represent the mean ± SD (*p<0.05, **p<0.01, compared with the control) unpaired Student’s t test.
FIGURES2.png
Fig S2. Effect of LPS on gene expression in BMECs. (A) Heatmap showing RNA sequencing data between control and LPS-treated BMECs, n = 3 for each group. (B) KEGG analysis of significantly regulated genes (P < 0.05). (C) Upregulated (red) and downregulated (blue) GO analysis of significantly regulated genes (P < 0.05) comparing LPS treatment and control. (D) Heatmap of the top 50 differentially expressed genes after LPS treatment in BMECs.
FIGURES3.png
Fig S3. Tight junction expression of cow tissue. (A - C) Immunohistochemical staining of ZO-1, Occludin, and Claudin-3 in the mammary glands of normal and mastitic dairy cows (n=3). Scale bar=100 μm.
FIGURES4.png
Fig S4. The optimal concentration of the TGFβR1-specific inhibitor SB431542 was screened (A - B) qPCR analysis of TGFβR1, Smad2/3 and Msi2 expression in BMECs after treatment with different concentrations of SB431542. The data represent mean ± SD, n = 3, *p<0.05, unpaired Student t test. (C) qPCR results showing Msi2 expression in BMECs after pcDNA-Msi2 transfection and SB431542 treatment. The data represent mean ± SD, **p<0.01, ****p<0.0001, unpaired Student t test.
FIGURES5.png
Fig S5. Blood routine and organ coefficients in the mastitis mouse model. (A) Blood biochemical indicators of the mastitis mouse model. WBC: White blood cell count. NE: neutrophilic granulocyte. LY: Lymphocytes. MO: monocytes. The data represent mean ± SD, **p<0.01, unpaired Student t test. (B) The ratio of NE, LY, and MO relative to WBCs in mouse blood. (C) The organ coefficient in mice. The data represent mean ± SD, n=3, *p<0.05, **p<0.01, unpaired Student t test. (D) qPCR analysis of NLRP3 expression in mice. The data represent mean ± SD, n = 3, **p<0.01, unpaired Student t test.
SupplementaryTable.docx
Supplementarymethods.docx

Download PDF

Version 1

posted

You are reading this latest preprint version

Musashi2 binds TGFβR1 regulates mastitis via TGFβ/Smad signaling pathway in dairy cows

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Animals

Animal experiments

Cell culture

RNAseq

Real-time fluorescent quantitative PCR

Western blotting

ELISA

Myeloperoxidase (MPO) activity assay

Immunofluorescence and histopathology

Cellular immunofluorescence

RNA-binding protein immunoprecipitation

Statistical analysis

Results

Discussion

Conclusions

Abbreviations

Declarations

References

Supplementary Files

Status:

Version 1