*2.6. Fetuin-A Inhibits Autophagic Flux*

Autophagy promotes primary cilium formation under serum deprivation. Therefore, we examined whether fetuin-A affects autophagy. First, we examined the autophagy by immunostaining with an antibody against LC3. In the control cells, the LC3 signal was hardly detected, however, Fetuin-A treatment induced LC3 accumulation in the cytoplasm, suggesting that fetuin-A treatment affected autophagy (Figure 7A). The accumulation of LC3 signal in the cytoplasm might result from the

*2.6. Fetuin-A Inhibits Autophagic Flux*

acceleration of autophagy or defective autophagic flux, and thus the conversion of LC3 I to LC3 II was examined by immunoblotting assay. Upon fetuin-A treatment, the conversion of LC3 I to LC3 II was reduced, as shown by a lowered LC3 II to I ratio (Figure 7B, *p* = 0.02), suggesting that the autophagic flux was reduced. To further confirm this observation, the expression of Beclin1, the key enzyme in the initiation of autophagic flux, was examined. The expression of Beclin1 was reduced by fetuin-A treatment (Figure 7C). Thus, fetuin-A inhibits autophagic flux. II was examined by immunoblotting assay. Upon fetuin-A treatment, the conversion of LC3 I to LC3 II was reduced, as shown by a lowered LC3 II to I ratio (Figure 7B, *p* = 0.02), suggesting that the autophagic flux was reduced. To further confirm this observation, the expression of Beclin1, the key enzyme in the initiation of autophagic flux, was examined. The expression of Beclin1 was reduced by fetuin-A treatment (Figure 7C). Thus, fetuin-A inhibits autophagic flux.

the acceleration of autophagy or defective autophagic flux, and thus the conversion of LC3 I to LC3

*Int. J. Mol. Sci.* **2019**, *20*, x FOR PEER REVIEW 9 of 13

results are the mean ± SD from three independent experiments, more than 100 cells were counted in

Autophagy promotes primary cilium formation under serum deprivation. Therefore, we examined whether fetuin-A affects autophagy. First, we examined the autophagy by immunostaining with an antibody against LC3. In the control cells, the LC3 signal was hardly detected, however,

each individual group. DNA was stained with DAPI. Scale bar 10 µM.

**Figure 7.** Fetuin-A inhibits autophagy. (**A**) Autophagy was examined by immunostaining with an antibody against LC3. DNA was stained with DAPI. Scale bar 10 µM. (**B**,**C**) Extracts of control (CTL) or fetuin-A (FA)-treated cells were analyzed by immunoblotting with antibodies against LC3, Beclin1, or actin. (**B**, lower panel) Quantification results of the relative intensity of the LC3 II to LC3 I ratio. \* **Figure 7.** Fetuin-A inhibits autophagy. (**A**) Autophagy was examined by immunostaining with an antibody against LC3. DNA was stained with DAPI. Scale bar 10 <sup>µ</sup>M. (**B**,**<sup>C</sup>**) Extracts of control (CTL) orfetuin-A (FA)-treated cells were analyzed by immunoblotting with antibodies against LC3, Beclin1, or actin. (**B**, lower panel) Quantification results of the relative intensity of the LC3 II to LC3 I ratio. \* *p* < 0.05.

#### *p* < 0.05. **3. Discussion**

**3. Discussion** In this study, we demonstrated that fetuin-A is relevant for cell growth and ciliogenesis in the placentas of GDM patients. Treatment with fetuin-A leads to abnormal centrosome amplification followed by the induction of aberrant mitotic spindle poles. In addition, fetuin-A also disorganizes the microtubule arrays. Furthermore, fetuin-A inhibits autophagy, followed by the reduction of primary cilium formation in placental cells. Taken together, fetuin-A affects the centrosome and In this study, we demonstrated that fetuin-A is relevant for cell growth and ciliogenesis in the placentas of GDM patients. Treatment with fetuin-A leads to abnormal centrosome amplification followed by the induction of aberrant mitotic spindle poles. In addition, fetuin-A also disorganizes the microtubule arrays. Furthermore, fetuin-A inhibits autophagy, followed by the reduction of primary cilium formation in placental cells. Taken together, fetuin-A affects the centrosome and autophagy, leading to defective placenta growth.

autophagy, leading to defective placenta growth. Circulating fetuin-A is upregulated in GDM patients [26], however, little is known about the effect of fetuin-A on the placenta. In this study, we showed that the placental fetuin-A concentration increases in GDM women. It is an important issue to clarify the source of placental fetuin-A. The elevated fetuin-A might be derived from maternal circulation. Additionally, it might also be synthesized locally at the placenta. In our study, we found that the mRNA level of fetuin-A increased in the placentas of GDM patients. In addition, a high glucose concentration induced the expression of fetuin-A in immortalized placental cells, suggesting that fetuin-A was also synthesized locally. Thus, fetuin-A might act on the placenta through paracrine or autocrine effects. Circulating maternal fetuin-A acts on the placenta via a paracrine effect. A high maternal glucose concentration also induces placental

cells to produce more fetuin-A locally. Thus, both circulating and local placental fetuin-A coordinately affect placental development in GDM.

The primary cilium regulates development and differentiation and, additionally, this tiny organelle plays roles in female reproduction. Endocrine gland-derived vascular endothelial growth factor (EG-VEGF), the key endocrine factor for proper placentation, binds to its receptor on the primary cilium, triggering the downstream signaling cascade for trophoblast invasion [23,27]. The regulation of ciliogenesis is initiated by TTBK2 recruitment and CP110 removal from the mother centriole [28], and resorption of the primary cilium is triggered by HDAC6 activation [29]. Recent studies showed that the primary cilia is also regulated by the miR-200 family in the placenta [27]. In this sstudy, we showed that fetuin-A also inhibits ciliogenesis in placental cells. So far, it is still unclear how fetuin-A affects the placental cilia. The concentration of miR-200 family members is upregulated in women with GDM [30]. In addition, the concentrations of fetuin-A and miR-200 family members increase in nonalcoholic fatty liver disease patients [10,31]. Thus, we speculate that, in the placentas of GDM women, the fetuin-A might induce the expression of miR-200 family members, thus inhibiting the formation of the primary cilia, however, this hypothesis still needs to be tested.

In summary, we showed that fetuin-A inhibits placental cell growth and is required for the control of centrosome homeostasis and ciliogenesis, which are two important events for proper placentation.
