Bmp2 regulates Serpinb6b expression via cAMP/PKA/Wnt4 pathway during uterine decidualization

Abstract Serpinb6b is a novel member of Serpinb family and found in germ and somatic cells of mouse gonads, but its physiological function in uterine decidualization remains unclear. The present study revealed that abundant Serpinb6b was noted in decidual cells, and advanced the proliferation and differentiation of stromal cells, indicating a creative role of Serpinb6b in uterine decidualization. Further analysis found that Serpinb6b modulated the expression of Mmp2 and Mmp9. Meanwhile, Serpinb6b was identified as a target of Bmp2 regulation in stromal differentiation. Treatment with rBmp2 resulted in an accumulation of intracellular cAMP level whose function in this differentiation program was mediated by Serpinb6b. Addition of PKA inhibitor H89 impeded the Bmp2 induction of Serpinb6b, whereas 8‐Br‐cAMP rescued the defect of Serpinb6b expression elicited by Bmp2 knock‐down. Attenuation of Serpinb6b greatly reduced the induction of constitutive Wnt4 activation on stromal cell differentiation. By contrast, overexpression of Serpinb6b prevented this inhibition of differentiation process by Wnt4 siRNA. Moreover, blockage of Wnt4 abrogated the up‐regulation of cAMP on Serpinb6b. Collectively, Serpinb6b mediates uterine decidualization via Mmp2/9 in response to Bmp2/cAMP/PKA/Wnt4 pathway.


K E Y W O R D S
Bmp2, cAMP-PKA-Wnt4 pathway, decidualization, Serpinb6b, uterine stromal cell program. 5,7 Further analysis found that physiological function of Bmp2 in decidualization was mediated by wingless-related murine mammary tumour virus integration site 4 (Wnt4). 7,8 However, the downstream regulatory mechanisms by which Bmp2-Wnt4 pathway controls stromal differentiation remain to be further elucidated.
Serine peptidase inhibitors (Serpins) are the largest and most broadly distributed family of protease inhibitors and essential for regulating diverse biological processes, including immune function, apoptosis, blood clotting, tumorigenesis and cancer metastasis. 9 Serpins in clade B consists of intracellular serpins, and its physiological functions have been widely studied in programmed cell death, autoimmunity, inflammatory and cancer progression. [10][11][12][13][14][15] Serpin, clade B, member 6b (Serpinb6b, also named as serine protease inhibitor), has been identified as a novel member of Serpinb family. Only report demonstrated that Serpinb6b was expressed in germ and somatic cells of mouse gonads, 16 but there is almost no research to elucidate its biological function. Based on the preliminary microarray analysis, 17 Serpinb6b is up-regulated in polyploid decidual cells as compared to non-polyploid cells, implying an involvement of Serpinb6b in decidualization.
The present study uncovers a novel insight into the role of Serpinb6b in uterine decidualization and identifies it as a downstream target of cAMP/PKA/Wnt4 pathway in response to Bmp2.

| Ethics approval
All animal experiment procedures were approved by the Committee for the Ethics on Animal Care and Use of Jilin University (SY201905031).

| Animal treatment
Matured Kunming white mice were caged in a controlled environment with a cycle of 14L:10D. Adult female mice were mated with fertile or vasectomized males to induce pregnancy or pseudopregnancy (day 1 = day of vaginal plug). Model of artificial decidualization was induced by infusing 25 µL of sesame oil into one uterine horn on day 4 of pseudopregnancy, while the contralateral uninjected horn served as a control. The uteri were collected at 48, 72 or 96 hours after oil infusion.

| In situ hybridization
In situ hybridization was performed as described previously. 18 Briefly, cRNA probes were labelled with Digoxigenin according to RNA labelling kit (Roche). After fixation and hybridization with above probes, frozen sections were incubated with sheep anti-digoxigenin antibody conjugated to alkaline phosphatase (1:5000; Roche) and then stained with 5-bromo-4-chloro-3-indolyl phosphate (BCIP, 0.4 mmol/L)/nitroblue tetrazolium (NBT, 0.4 mmol/L) buffer. The sense probe was also hybridized and served as a negative control.
Controls received the vehicle only.

| RNA interference
Stromal cells were transfected with siRNAs using Lipofectamine™ 3000 (Invitrogen). After transfection with Alk2, Bmp2, Wnt4 or Serpinb6b siRNA, cells were collected at 48 hours in the absence or presence of rBmp2 or 8-Br-cAMP. The corresponding siRNA sequences were list in Table 2.

| Plasmid construction and transfection
Full-length cDNA fragments were amplified using the following prim-

| Cell proliferation
After transfection with Serpinb6b overexpression plasmid or siRNA, stromal cells were cultured for 48 hours, added MTS reagent (Promega) into each well and then incubated for another 2-3 hours.
Absorbance was measured at 490 nm using a multi-mode reader.

| Intracellular cAMP measurement
The intracellular cAMP levels were measured as described previously. 19 After transfection with Alk2 siRNA and addition of rBmp2, stromal cells were cultured for 48 hours. After removing the media, cells were incubated with the Induction Buffer containing 100 μmol/L 4-(3-butoxy-4-methoxy-benzyl) imidazolidone and 500 μmol/L IBMX, and then tested using cAMP-Glo™ Assay (Promega). Luminescence was measured with a Cell Imaging Multi-Mode Reader.

| Statistics
All the experiments were independently repeated at least three times. Comparison of two groups was made by independent samples t test. And the multiple comparisons were tested with one-way ANOVA. Both statistical analyses were performed using SPSS19.0 software program (SPSS Inc). P < .05 was considered statistically significant.

| Serpinb6b mRNA expression during decidualization
To explore the correlation between Serpinb6b and uterine decidualization, we examined its expression in the uteri during early pregnancy and artificial decidualization. The results showed that Serpinb6b mRNA signal was extremely faint in the uterus from days 1 to 5 of pregnancy ( Figure 1B When Serpinb6b antisense probe was replaced by its sense probe, there was no detectable signal in day 8 uterus ( Figure 1A). Real-time PCR analysis also exhibited an expected elevation on days 6 to 8 of

TA B L E 2
The siRNAs used in this study pregnancy ( Figure S1A). Meanwhile, a high level of Serpinb6b mRNA signal was seen in the decidualizing stromal cells under artificial decidualization, while no signal was found in the uninjected control uteri (Figure 2A-F). Consistently, real-time PCR result revealed that Serpinb6b expression was time-dependently up-regulated followed by oil infusion ( Figure S1B).

| Role of Serpinb6b during decidualization
To determine the function of Serpinb6b in uterine decidualization, we analysed its influences on stromal cell proliferation and differentiation which are two crucial steps for decidualization. 2 After transfection with Serpinb6b overexpression plasmid which significantly up-regulated its mRNA level, proliferation activity of stromal cells was obviously enhanced ( Figure 3A

| Regulation of Serpinb6b on the expression of Mmp2 and Mmp9
To explore a potential mechanism for Serpinb6b in uterine decidualization, we analysed its regulation on the expression of matrix metallopeptidase 2 (Mmp2) and Mmp9 whose expression in the decidua was overlapped with that of Serpinb6b. 21 The results showed that after introduction of Serpinb6b overexpression plasmid, stromal cells exhibited obvious elevation for mRNA levels of Mmp2 and Mmp9, while silencing of Serpinb6b repressed their expression ( Figure 3G,H).

| Bmp2 regulation of Serpinb6b expression
As Bmp2 is intensively expressed in uterine decidua of pregnant mice and has been demonstrated to be a key regulator of decidualization, [5][6][7][8]22 we studied the relevance between Bmp2 and Serpinb6b.
After exposure to rBmp2, stromal differentiation was induced followed by a time-dependent elevation for Serpinb6b expression ( Figure 4A; Figure S2A). Moreover, neither overexpression nor silencing of Serpinb6b had obvious effects on Bmp2 expression ( Figure 4B). When activin-like kinase 2 (Alk2), a Bmp type I receptor, was silenced by specific siRNA, the up-regulation of rBmp2 on Serpinb6b expression was significantly weakened, accompanying with reduced mRNA levels of Prl8a2 and Prl3c1 ( Figure 4C).
By contrast, knock-down of Bmp2 had the opposite effectiveness ( Figure 4D; Figure S2B). To verify whether Serpinb6b was involved in Bmp2 function in stromal differentiation, we transfected the stromal cells with Serpinb6b siRNA, then added rBmp2 and analysed the expression of Prl8a2 and Prl3c1. The results showed that attenuation of Serpinb6b expression greatly reduced the rBmp2 induction of Prla8a2 and Prl3c1 ( Figure 4E). Conversely, overexpression of Serpinb6b evidently reversed the suppression of Prl8a2 and Prl3c1 by Bmp2 siRNA ( Figure 4F).

| Serpinb6b mediated the effects of cAMP on stromal cell differentiation
Activation of cAMP-PKA signalling is vital for induction of decidualization. 23,24 In uterine stromal cells, Serpinb6b expression was

| Bmp2 regulates Serpinb6b expression via cAMP-PKA signalling
In stromal cells, 8-Br-cAMP did not adjust Bmp2 expression with or without PKA inhibitor H89 ( Figure 5E,F). However, supplementation of exogenous rBmp2 resulted in an obvious accumulation of intracellular cAMP level, but this accumulation was reversed by Alk2 siRNA ( Figure 5G). In the meantime, H89 impeded the induction of stromal Bmp2 is upstream of cAMP-PKA signalling ( Figure 5H,I). As demonstrated above, Serpinb6b was regulated by cAMP via PKA signalling. We next dissected whether Bmp2 regulation of Serpinb6b was mediated by cAMP-PKA signalling. After pre-treatment with H89, the induction of rBmp2 on Serpinb6b was remarkably blocked ( Figure 5H). In contrast, 8-Br-cAMP ameliorated the reduction of Serpinb6b generated by Bmp2 knock-down ( Figure 5I).

| Wnt4 mediates the regulation of cAMP on Serpinb6b expression
Wnt4 is important for uterine decidualization.  Figure 6F).

| Bmp2 modulates Serpinb6b expression through cAMP/PKA/Wnt4 pathway
After supplementation of exogenous rBmp2, Wnt4 induction was noted in stromal cells, but this induction was hindered by Alk2 siRNA ( Figure 7A). Both overexpression and inhibition of Wnt4 did not change the expression of Bmp2 in the stromal cells ( Figure 7B).
As reported previously, Alk2 induced the accumulation of intracellular cAMP level. 19 Addition of 8-Br-cAMP rescued the suppression of Bmp2 siRNA on Wnt4 expression, whereas PKA inhibitor H89 treatment led to a failure in the stimulation of rBmp2 on Wnt4 ( Figure 7C,D).
As described above, Bmp2 regulated the expression of Serpinb6b through cAMP-PKA signalling which was upstream of Wnt4. We

| D ISCUSS I ON
Serpinb6b is a novel identified member of Serpinb family, but its biology function remains unclear although was found in germ and somatic cells of mouse gonads. 16 The present study revealed that abundant Serpinb6b was noted in decidual cells, which confirmed the previous microarray data, 17 suggesting an involvement of Serpinb6b in uterine decidualization.
It has been established that uterine decidualization is characterized by extensive proliferation and differentiation of stromal cells in mice. 2 Here, Serpinb6b could enhance the proliferation activity of uterine stromal cells. It is well known that cyclins and Cdks are the main regulator of mammalian cell proliferation. 26 Ccna1 has been demonstrated to be involved in the cell cycle progression from S to G2 phase. 26 Among the numerous Cdks, Cdk1 is truly essential for mammalian cell cycle because disruption of Cdk1 is embryonic lethal at blastocyst stage, while mice deficiency of Cdk2, Cdk3, Cdk4 or Cdk6 are viable. 27,28 Previous studies reported that Ccna1 was able to complex with Cdk1 to drive the S to G2 phase transition and also coordinate with Cdk2 to facilitate cell entry into the S phase. 29,30 In this study, Serpinb6b could induce the expression of Ccna1 and Cdk1, but had no effect on the expression of Cdk2, implying that Serpinb6b is involved in regulating cell cycle progression from S into G2 phase. In conclusion, the present study revealed a novel insight into the role of Serpinb6b in uterine decidualization via Mmp2/9 and identified it as a downstream target of Bmp2/cAMP/PKA/Wnt4 pathway ( Figure 8).