Transcriptional regulation of cyclophilin D by BMP/Smad signaling and its role in osteogenic differentiation
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Evaluation Summary:
This paper is of potential interest to investigators in the bone field. The study provides evidence of CypD regulation of osteoblast differentiation and offers new insights into it's transcriptional regulation. Overall, although the findings are compelling and have the potential to advance the field, several conclusions require additional data or clarification and there are some missed opportunities to strengthen the manuscript.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)
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Abstract
Cyclophilin D (CypD) promotes opening of the mitochondrial permeability transition pore (MPTP) which plays a key role in both cell physiology and pathology. It is, therefore, beneficial for cells to tightly regulate CypD and MPTP but little is known about such regulation. We have reported before that CypD is downregulated and MPTP deactivated during differentiation in various tissues. Herein, we identify BMP/Smad signaling, a major driver of differentiation, as a transcriptional regulator of the CypD gene, Ppif . Using osteogenic induction of mesenchymal lineage cells as a BMP/Smad activation-dependent differentiation model, we show that CypD is in fact transcriptionally repressed during this process. The importance of such CypD downregulation is evidenced by the negative effect of CypD ‘rescue’ via gain-of-function on osteogenesis both in vitro and in a mouse model. In sum, we characterized BMP/Smad signaling as a regulator of CypD expression and elucidated the role of CypD downregulation during cell differentiation.
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Evaluation Summary:
This paper is of potential interest to investigators in the bone field. The study provides evidence of CypD regulation of osteoblast differentiation and offers new insights into it's transcriptional regulation. Overall, although the findings are compelling and have the potential to advance the field, several conclusions require additional data or clarification and there are some missed opportunities to strengthen the manuscript.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
This manuscript investigates CypD gene regulation in OB differentiation. CypD regulates MPTP opening in IMM and hence its significance in regulating Mt function, OxPhos and cell differentiation.
The data demonstrate presence of potential Smad-binding elements in the upstream region of Ppif gene and functionally confirmed by down regulation of CypD mRNA expression in the presence of Smad1 over expression, while Runx2 expression was induced. Using ChIP assay the authors then attempted to demonstrate interaction of Smad1 with Ppif promoter in response to BMP and it's functionality using appropriate Luc reporter construct. Additional inhibitory approaches such as Smad7 and noggin were considered to validate this response. The data further show that Ppif gene expression and CypD protein expression are reduced …
Reviewer #1 (Public Review):
This manuscript investigates CypD gene regulation in OB differentiation. CypD regulates MPTP opening in IMM and hence its significance in regulating Mt function, OxPhos and cell differentiation.
The data demonstrate presence of potential Smad-binding elements in the upstream region of Ppif gene and functionally confirmed by down regulation of CypD mRNA expression in the presence of Smad1 over expression, while Runx2 expression was induced. Using ChIP assay the authors then attempted to demonstrate interaction of Smad1 with Ppif promoter in response to BMP and it's functionality using appropriate Luc reporter construct. Additional inhibitory approaches such as Smad7 and noggin were considered to validate this response. The data further show that Ppif gene expression and CypD protein expression are reduced during osteogenic differentiation. Conversely, expression of caCypD inhibited osteogenesis.
Overall, the findings of this study shed some additional light on the transcriptional activity of Ppif in osteoblasts, yet only moderately advance current knowledge related to the role of CypD as a regulator of osteogenesis. Specifically, the role of CypD in osteogenesis has been documented demonstrating benefits of CypD loss of function to bone. Hence, demonstrating the negative impact of CypD gain of function on osteogenesis and bone formation is only confirmatory and appears to not advance current knowledge. However, although certain aspects of the transcriptional regulation have been investigated, this study adds additional important insights. -
Reviewer #2 (Public Review):
Eliseev and colleagues investigated the hypothesis that BMP signaling indirectly regulates the function of the mitochondrial permeability transition pore by controlling the transcription of cyclophilin D, a component of the pore. As mitochondria dysfunction correlates with increased pathophysiology in many diseases, understanding how mitochondrial function is regulated is of significant interest. Here the authors use the skeleton as a model system, and employ a variety of methodologies, including mouse loss and gain of function studies, cell based assays, bulk RNAseq, and phenotype rescue experiments, to examine the relationship between BMP signaling, cyclophilin D expression and bone formation. This experimental approach produced data that convincing demonstrates that BMP signaling can inhibit opening of …
Reviewer #2 (Public Review):
Eliseev and colleagues investigated the hypothesis that BMP signaling indirectly regulates the function of the mitochondrial permeability transition pore by controlling the transcription of cyclophilin D, a component of the pore. As mitochondria dysfunction correlates with increased pathophysiology in many diseases, understanding how mitochondrial function is regulated is of significant interest. Here the authors use the skeleton as a model system, and employ a variety of methodologies, including mouse loss and gain of function studies, cell based assays, bulk RNAseq, and phenotype rescue experiments, to examine the relationship between BMP signaling, cyclophilin D expression and bone formation. This experimental approach produced data that convincing demonstrates that BMP signaling can inhibit opening of the mitochondrial permeability transition pore to regulate the function of bone forming cells and ultimately enhance bone formation. However, in several instances the data provided do not completely support the authors' conclusions, and the specific impact on the study of age-related bone loss still remains to be determined. An additional significance of this work is the ability to test if the regulation of the mitochondrial permeability transition pore by BMP signaling is a general phenomenon of cell physiology. As BMP signaling occurs in all cell types, and this proposed function has not been previously studies, the authors data support a conceptual advance in our understanding of how the many distinct BMPs exert control over biological functions.
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Reviewer #3 (Public Review):
The TGF-beta superfamily BMP/Smad transcription factors play a pivotal role in multiple pathophysiological processes including bone morphogenesis, remodeling. This study attempts to show that CypD (Ppif) gene is an important target of BMP/Smad. In particular, the authors stress that down regulation of CypD gene expression by overactivation of BMP/Smad negatively regulates the transcription of Ppif gene thereby suppressing MPTP, a process which leads to cell death. The study also suggests that the pathway becomes defective in aging which may be the cause of bone defects.
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