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KLF2 Regulates Neural Differentiation of Dental Pulp-derived Stem Cells by Modulating Autophagy and Mitophagy

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Abstract

Background

Transplantation of stem cells for treating neurodegenerative disorders is a promising future therapeutic approach. However, the molecular mechanism underlying the neuronal differentiation of dental pulp-derived stem cells (DPSC) remains inadequately explored. The current study aims to define the regulatory role of KLF2 (Kruppel-like factor 2) during the neural differentiation (ND) of DPSC.

Methods

We first investigated the transcriptional and translational expression of KLF2, autophagy, and mitophagy-associated markers during the ND of DPSC by using quantitative RT-PCR and western blot methods. After that, we applied the chemical-mediated loss- and gain-of-function approaches using KLF2 inhibitor, GGPP (geranylgeranyl pyrophosphate), and KLF2 activator, GGTI-298 (geranylgeranyl transferase inhibitor-298) to delineate the role of KLF2 during ND of DPSC. The western blot, qRT-PCR, and immunocytochemistry were performed to determine the molecular changes during ND after KLF2 deficiency and KLF2 sufficiency. We also analyzed the oxygen consumption rate (OCR) and the extracellular acidification rate (ECAR) using the Seahorse XFe24 analyzer.

Results

Our study demonstrated that the expression level of KLF2, autophagy, and mitophagy-associated markers were significantly elevated during the ND of DPSC. Next, we found that the KLF2 inhibitor, GGPP significantly reduced the ND of DPSC. Inversely, KLF2 overexpression accelerated the molecular phenomenon of DPSC’s commitment towards ND, indicating the crucial role of KLF2 in neurogenesis. Moreover, we found that the KLF2 positively regulated autophagy, mitophagy, and the Wnt5a signaling pathway during neurogenesis. Seahorse XFe24 analysis revealed that the ECAR and OCR parameters were significantly increased during ND, and inhibition of KLF2 marginally reversed them towards DPSC’s cellular bioenergetics. However, KLF2 overexpression shifted the cellular energy metabolism toward the quiescent stage.

Conclusion

Collectively, our findings provide the first evidence that the KLF2 critically regulates the neurogenesis of DPSC by inducing autophagy and mitophagy.

Graphical Abstract

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Data Availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Abbreviations

CNS:

Central nervous system

DPSC:

Dental pulp-derived stem cells

ECAR:

Extracellular acidification rate

GGPP:

Geranylgeranyl pyrophosphate

GGTI-298:

Geranylgeranyl transferase inhibitor-298

HBSS:

Hanks’ balanced salt solution

KLF2:

Krüppel-like factor 2

KLFs:

Krüppel-like factors

MEME:

Minimum essential medium eagle

MSCs:

Mesenchymal stem cells

ND:

Neural differentiation

NDM:

Neural differentiation medium

OCR:

Oxygen consumption rate

ROS:

Reactive oxygen species

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Acknowledgements

We are thankful to Zijuan Liu, from TTUHSC core facility for her help in procuring some of the confocal images. The core facility is being supported by a Core Facility Support Award (grant number RP200572) from the Cancer Prevention and Research Institute of Texas (CPRIT) to the Imaging Core, Texas Tech University Health Sciences Center at Amarillo.

Funding

This work was supported in part by National Institutes of Health grants, R01AR068279 (NIAMS), STTR R42EY031196 (NEI), and STTR 1R41AG057242 (NIA). The funders had no role in the study design, data collection, and analysis, decision to publish, or preparation of the manuscript.

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  1. Department of Pharmaceutical Sciences, Jerry H. Hodge School of Pharmacy, Texas Tech University Health Sciences Center, ARB Suite 2116, 1406 South Coulter Street, Amarillo, TX, 79106, USA

    Prateeksha Prateeksha, Prathyusha Naidu, Manjusri Das, Derek Barthels & Hiranmoy Das

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  1. Prateeksha Prateeksha
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All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Study conception, design, and manuscript writing: PP and HD. Acquisition of data: PP, PN, MD, DB Analysis and interpretation of data: PP and HD.

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Correspondence to Hiranmoy Das.

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Prateeksha, P., Naidu, P., Das, M. et al. KLF2 Regulates Neural Differentiation of Dental Pulp-derived Stem Cells by Modulating Autophagy and Mitophagy. Stem Cell Rev and Rep 19, 2886–2900 (2023). https://doi.org/10.1007/s12015-023-10607-0

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