Elucidation of the role for Krüppel-like factor 4 & its action mechanism in the follicular-luteal shift in ovarian steroidogenesis

Abstract

The preovulatory luteinizing hormone (LH) surge induces differentiation of the follicular cells to luteal cells, and is accompanied by a dramatic reduction in Cyp19A1, which leads to a rapid shift in steroidogenesis from estradiol to progesterone. However, the mechanism underlying the inhibition of Cyp19A1 expression after the LH surge is not well understood.

Krüppel-like factor 4 (Klf4) has been shown to play a role in regulating the cell differentiation and cell cycle control in various cell types. In this study, I investigated the regulatory mechanisms of Klf4 expression and its role during luteinization process by LH/human chorionic gonadotropin (hCG) using in vivo and in vitro rat experimental models. Klf4 expression increased rapidly within an hour and then sharply decreased after LH/hCG treatment of preovulatory follicles in vivo and in vitro by real time RT-PCR, western blot, and immunohistochemistry.

Treatments with LH or forskolin stimulated Klf4 mRNA expression in granulosa cells (GCs) and this stimulatory effects were reduced by inhibitors of protein kinase A (PKA), indicating that Klf4 expression was mediated through the activation of the PKA signaling pathway. To evaluate the transcriptional regulation of Klf4 by LH, Klf4 promoter deletion constructs were generated. Luciferase reporter assays revealed that the -715/-402 region was involved in LH-stimulation of Klf4 promoter activity. Sp1 overexpression or inhibition of Sp1 binding revealed that Sp1 and its binding sites were important in LH-induced Klf4 regulation. In addition, Chromatin immunoprecipitation (ChIP) assays verified the in vivo binding of Sp1 to the Klf4 promoter region (-715/-402). Luciferase reporter assays showed that mutation of the -698 Sp1 binding site markedly decreased the basal and LH-induced Klf4 promoter activity.

Since the mechanism underlying the inhibition of Cyp19A1 expression during GCs differentiation is not well understood, I analyzed whether Klf4 is involved in the rapid decline of Cyp19A1 in GCs. Cyp19A1 expression in GCs transfected with FLAG-Klf4 or Klf4-specific siRNA was analyzed by real-time RT-PCR and immunofluorescence staining. Cyp19A1 decreased when Klf4 was overexpressed, and Cyp19A1 and estradiol biosynthesis increased when Klf4 was knocked down. The mechanism by which Klf4 regulates Cyp19A1 expression was investigated using Cyp19A1 promoter-luciferase reporter assays and ChIP assays. The results

revealed that the SF1-binding motif, but not the Sp1 binding element or the CACCC motif, was required for Klf4-mediated repression of Cyp19A1 promoter activity.

After LH surge occurs, cell proliferation stops and the cell cycle is arrested. To analyze whether Klf4 affects cell cycle-related genes, in which GCs were transfected with a FLAG-Klf4 or Klf4-specific siRNA, followed by determination of the transcript levels of cell cycle-related genes. The transcript levels of cell cycle promoters (Cyclin D1 and Cyclin D2) decreased, whereas those of a cell cycle inhibitor, p21 Cip1

, increased. Altering expression of Klf4 by overexpression or knockdown consistently affected the expression of Cyclin D2. In agreement with this, Klf4 overexpression reduced cell proliferation and arrested cell cycle

progression in G0/G1 phase.

Taken together, this study confirmed that Klf4 was activated by LH surge via PKA signaling pathway and mediated by Sp1 and Sp1 binding motif ( -698 Sp1) in preovulatory GCs. Functionally, Klf4 suppressed Cyp19A1 expression by directly binding to the SF1 binding motif in the Cyp19A1 promoter. Klf4 terminated GCs

proliferation and induced differentiation by regulating cell cycle-related genes.

These findings suggest that Klf4 is a key regulator induced by LH surge that has an essential role in the luteal transition in steroidogenesis as well as cell cycle regulation.|Krüppel-like factor 4 (Klf4)는 여러 세포의 증식 및 억제, 분화, 세포주기 등에 관련되어 있다고 알려진 Zinc finger 전사인자이다. 성선자극호르몬은 난소에서 배란을 유도하고, estrogen을 생산하는 과립막세포가 progesterone을 생산하는 황체세포로 변환하는데 중요한 역할을 하며, 또한 Klf4의 발현도 조절한다고 보고되었다. 이 연구에서는 난소의 과립막세포가 황체화세포로 분화되는 과정 동안, Klf4의 발현 여부와 발현 위치를 확인하고 성선자극호르몬에 의한 Klf4의 조절경로 및 Klf4가 steroid 호르몬 분비변화 및 세포주기에 미치는 영향을 분석하고자 하였다.

성선자극호르몬을 투여한 미성숙 쥐의 난소와 과립막세포에서 시간에 따른 Klf4의 mRNA와 protein의 발현변화를 Real time RT-PCR과 Western blot으로 확인하고, 쥐의 난소 내에서 Klf4의 발현부위 및 양상을 Immunohistochemistry를 이용하여 조사한 결과, LH 처리 후 1시간 이내에 난소와 과립막세포에서 모두 Klf4 발현이 급격히 증가한 후 감소하는 것을 확인하였다. Klf4는 PKA signaling pathway를 통해서 발현 증가하는 것을 확인하였으며, Klf4 promoter에서 어느 부분이 LH에 반응해 활성이 증가하는지 Klf4 promoter deletion construct를 제작하여 확인한 결과, Klf4 promoter 활성증가에 –715/-402 region이 중요한 것을 확인하였다. LH surge에 의해 Sp1 activity가 증가하고 Sp1이 비생식세포에서 Klf4 발현을 조절한다고 알려져 있기 때문에, 과립막세포에서도 Sp1이 Klf4 promoter 활성 증가에 관여하는지 Luciferase assay로 분석하였고, ChIP assay를 통해 Sp1이 -715/-500에 위치하는 Sp1 binding site에 결합하는 것을 확인하였다. Klf4 promoter mutant constructs (-715/-500 bp)를 제작하여 Sp1 binding motif (-698Sp1) 가 중요하게 작용하는 것을 확인하였다.

LH surge후에 에스트로겐 합성에 중요한 Cyp19A1 발현이 감소되는데, 억제되는 기전에 대해서 밝혀진 바가 없기 때문에 분화의 중요한 마커로써 Cyp19A1를 타겟 유전자로 정해 연구를 진행하였다. 과립막세포에서 Klf4의 과발현 및 발현억제를 유도한 후 Cyp19A1 mRNA 발현변화를 Real time RT-PCR로 확인하고, 단백질 발현변화를 Immunocytochemistry로 분석하였다. ELISA를 이용하여 Klf4가 호르몬 변화에 영향을 미치는지 분석하고, 직접적으로 Klf4가 Cyp19A1 promoter 활성에 관여하는지 보기 위해서, 과립막세포에 Cyp19A1 promoter와 Klf4를 co-transfection 시켜서 Cyp19A1 promoter의 활성변화를 분석하였다. 실험결과, Klf4가 Cyp19A1 promoter에 작용하여 Cyp19A1 발현 감소와 연관이 있음을 확인하였고, estrogen 분비억제, progesterone 분비를 증가시킴으로써 스테로이드 호르몬 합성조절에 관여하는 것을 확인하였다. 반대로 Klf4 발현을 억제시킨 경우에는 Cyp19A1 발현과 배양액내 estradiol 농도가 증가하였다. Klf4가 Cyp19A1 promoter상 어느 부위에 작동하는지 분석하기 위해서, Klf4와 Sf1, Cyp19A1 promoter를 co-transfection 시킨 과립막세포에서 Sf1에 의해 Cyp19A1 promoter 활성이 증가하고 Klf4 농도가 증가함에 따라 프로모터 활성이 감소하는 것을 확인하고, ChIP assay를 통해서 Klf4가 Cyp19A1 promoter상의 SF1 binding site에 직접적으로 결합하여 Cyp19A1 발현억제에 관여한다는 것을 확인하였다.

과립막세포에서 LH dosage에 따른 세포주기 관련유전자들의 mRNA 발현변화를 확인 후, Klf4 과발현 및 발현억제를 유도 하였을 때, 세포주기와 관련된 유전자발현에 미치는 영향을 Real time RT-PCR로 확인한 결과, LH에 의해 positive cell cycle regulator인 Cyclin D1과 Cyclin D2는 발현감소, Cell cycle inhibitor인 p21Cip1은 발현이 증가하였다. Klf4 과발현에 의해서 세포증식과 생존율이 감소되는 것을 BrdU-incorporation assay와 CCK-8 assay로 확인하고 세포주기 G0/G1 단계에서 cell cycle arrest가 유도되는 것을 FACS analysis로 확인하였다.

따라서 본 연구결과, 난포 내 과립막세포가 LH surge에 의해 분화되는 과정에서 PKA 신호 전달 경로를 통해서 증가한 Klf4 활성에 전사인자 Sp1과 Klf4 promoter 상의 Sp1 binding motif (-698Sp1)가 Klf4 발현 조절에 관여하고, Klf4가 cell cycle arrest를 유도하고 과립막세포의 분화를 촉진하는데 estradiol 생산에 주요 효소인 Cyp19A1 promoter의 SF1 binding site에 직접적으로 작용하여 Cyp19A1의 발현 억제 및 estradiol 분비를 억제함으로써 난포의 황체가 시작되는 시점에서 중요한 역할을 하는 전사인자임을 확인하였다.; The preovulatory luteinizing hormone (LH) surge induces differentiation of the follicular cells to luteal cells, and is accompanied by a dramatic reduction in Cyp19A1, which leads to a rapid shift in steroidogenesis from estradiol to progesterone. However, the mechanism underlying the inhibition of Cyp19A1 expression after the LH surge is not well understood.

Krüppel-like factor 4 (Klf4) has been shown to play a role in regulating the cell differentiation and cell cycle control in various cell types. In this study, I investigated the regulatory mechanisms of Klf4 expression and its role during luteinization process by LH/human chorionic gonadotropin (hCG) using in vivo and in vitro rat experimental models. Klf4 expression increased rapidly within an hour and then sharply decreased after LH/hCG treatment of preovulatory follicles in vivo and in vitro by real time RT-PCR, western blot, and immunohistochemistry.

Treatments with LH or forskolin stimulated Klf4 mRNA expression in granulosa cells (GCs) and this stimulatory effects were reduced by inhibitors of protein kinase A (PKA), indicating that Klf4 expression was mediated through the activation of the PKA signaling pathway. To evaluate the transcriptional regulation of Klf4 by LH, Klf4 promoter deletion constructs were generated. Luciferase reporter assays revealed that the -715/-402 region was involved in LH-stimulation of Klf4 promoter activity. Sp1 overexpression or inhibition of Sp1 binding revealed that Sp1 and its binding sites were important in LH-induced Klf4 regulation. In addition, Chromatin immunoprecipitation (ChIP) assays verified the in vivo binding of Sp1 to the Klf4 promoter region (-715/-402). Luciferase reporter assays showed that mutation of the -698 Sp1 binding site markedly decreased the basal and LH-induced Klf4 promoter activity.

Since the mechanism underlying the inhibition of Cyp19A1 expression during GCs differentiation is not well understood, I analyzed whether Klf4 is involved in the rapid decline of Cyp19A1 in GCs. Cyp19A1 expression in GCs transfected with FLAG-Klf4 or Klf4-specific siRNA was analyzed by real-time RT-PCR and immunofluorescence staining. Cyp19A1 decreased when Klf4 was overexpressed, and Cyp19A1 and estradiol biosynthesis increased when Klf4 was knocked down. The mechanism by which Klf4 regulates Cyp19A1 expression was investigated using Cyp19A1 promoter-luciferase reporter assays and ChIP assays. The results

revealed that the SF1-binding motif, but not the Sp1 binding element or the CACCC motif, was required for Klf4-mediated repression of Cyp19A1 promoter activity.

After LH surge occurs, cell proliferation stops and the cell cycle is arrested. To analyze whether Klf4 affects cell cycle-related genes, in which GCs were transfected with a FLAG-Klf4 or Klf4-specific siRNA, followed by determination of the transcript levels of cell cycle-related genes. The transcript levels of cell cycle promoters (Cyclin D1 and Cyclin D2) decreased, whereas those of a cell cycle inhibitor, p21 Cip1

, increased. Altering expression of Klf4 by overexpression or knockdown consistently affected the expression of Cyclin D2. In agreement with this, Klf4 overexpression reduced cell proliferation and arrested cell cycle

progression in G0/G1 phase.

Taken together, this study confirmed that Klf4 was activated by LH surge via PKA signaling pathway and mediated by Sp1 and Sp1 binding motif ( -698 Sp1) in preovulatory GCs. Functionally, Klf4 suppressed Cyp19A1 expression by directly binding to the SF1 binding motif in the Cyp19A1 promoter. Klf4 terminated GCs

proliferation and induced differentiation by regulating cell cycle-related genes.

These findings suggest that Klf4 is a key regulator induced by LH surge that has an essential role in the luteal transition in steroidogenesis as well as cell cycle regulation.