Dataset Information

Phlpp1 alters the murine chondrocyte phospho-proteome during endochondral bone formation

ABSTRACT: Appendicular skeletal growth and bone mass acquisition are controlled by a variety of growth factors, hormones, and mechanical forces in a dynamic process called endochondral ossification. In long bones, chondrocytes in the growth plate proliferate and undergo hypertrophy to drive bone lengthening and mineralization. Pleckstrin homology (PH) domain and leucine rich repeat phosphatase 1 and 2 (Phlpp1 and Phlpp2) are serine/threonine protein phosphatases that regulate cell proliferation, survival, and maturation via Akt, PKC, Raf1, S6k, and other intracellular signaling cascades. Germline deletion of Phlpp1 suppresses bone lengthening in part through parathyroid hormone receptor-dependent signaling in growth plate chondrocytes. Here, we demonstrate that Phlpp2 does not regulate endochondral ossification, and we define the molecular differences between Phlpp1 and Phlpp2 in chondrocytes. Phlpp2-/- mice are phenotypically indistinguishable from their wildtype (WT) littermates, with similar bone length, bone mass, and growth plate dynamics. Deletion of Phlpp2 had moderate effects on the chondrocyte transcriptome and proteome compared to WT cells. By contrast, Phlpp1/2-/- (double knockout) mice resembled Phlpp1-/- mice phenotypically and chondrocyte phospho-proteomes of Phlpp1-/- and Phlpp1/2-/- chondrocytes were different than WT and Phlpp2-/- chondrocyte phospho-proteomes. Data integration via multiparametric analysis identified alterations in Pdpk1 and Pak1/2 signaling pathways in chondrocytes lacking Phlpp1. In conclusion, these data demonstrate that Phlpp1, but not Phlpp2, regulates endochondral ossification through multiple and complex signaling cascades.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Cell Culture

SUBMITTER: Benjamin Madden

LAB HEAD: Dr. Jennifer Westendorf Ph.D.

PROVIDER: PXD055563 | Pride | 2024-10-22

REPOSITORIES: Pride

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			Action	DRS
	Eclipse_2022Oct28_P22085_TotF01a.mgf.gz	Mgf
	Eclipse_2022Oct28_P22085_TotF01a.raw	Raw
	Eclipse_2022Oct28_P22085_TotF02.mgf.gz	Mgf
	Eclipse_2022Oct28_P22085_TotF02.raw	Raw
	Eclipse_2022Oct28_P22085_TotF03.mgf.gz	Mgf

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Phlpp1 alters the murine chondrocyte phospho-proteome during endochondral bone formation.

Weaver Samantha R SR Peralta-Herrera Eduardo E Torres Haydee M HM Jessen Erik E Bradley Elizabeth W EW Westendorf Jennifer J JJ

Bone 20240929

Appendicular skeletal growth and bone mass acquisition are controlled by a variety of growth factors, hormones, and mechanical forces in a dynamic process called endochondral ossification. In long bones, chondrocytes in the growth plate proliferate and undergo hypertrophy to drive bone lengthening and mineralization. Pleckstrin homology (PH) domain and leucine rich repeat phosphatase 1 and 2 (Phlpp1 and Phlpp2) are serine/threonine protein phosphatases that regulate cell proliferation, survival, ...[more]

PMID: 39349089

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Project description:Background: Glucocorticoids (GCs) are widely used anti-inflammatory drugs. While useful in clinical practice, patients taking GCs often suffer from skeletal side effects including growth retardation and decreased bone quality in adults. On a physiological level, GCs have been implicated in the regulation of chondrogenesis and osteoblast differentiation, as well as maintaining homeostasis in cartilage and bone. We identified the glucocorticoid receptor (GR) as a potential regulator of chondrocyte hypertrophy in a microarray screen of primary limb bud mesenchyme micromass cultures. Some targets of GC regulation in chondrogenesis are known, but the global effects of pharmacological GC doses on chondrocyte gene expression have not been comprehensively evaluated. Results: This study systematically identifies a spectrum of GC target genes in embryonic growth plate chondrocytes treated with synthetic GR agonist, dexamethasone (DEX), at 6 and 24 hrs. Conventional analysis of this data set and gene set enrichment analysis (GSEA) was performed. Transcripts associated with metabolism were enriched in the DEX condition along with extracellular matrix genes. In contrast, a subset of growth factors and cytokines were negatively correlated with DEX treatment. Comparing DEX-induced gene expression data to developmental changes in gene expression in micromass cultures revealed an additional layer of complexity in which DEX maintains the expression of certain chondrocyte marker genes while inhibiting factors that promote vascularization and ultimately ossification of the cartilaginous template. Conclusions: Together, these results provide insight into the mechanisms and major molecular classes functioning downstream of DEX in primary chondrocytes monolayers. In addition, comparison of our data with microarray studies of DEX treatment in other cell types demonstrated that the majority of DEX effects are tissue-specific. This study provides novel insights into the effects of pharmacological GC on chondrocyte gene transcription and establishes the basis for subsequent functional studies. Experiment Overall Design: Primary chondrocytes isolated from the tibiae, humeri and femurs of 15.5 day old mice are treated with 10EXP-7M DEX or an equal volume of the DMSO vehicle for 24 hrs. Total RNA is isolated after 6 hrs and 24 hrs of treatment. Samples from three independent experiments independent time courses were hybridized to Affymetrix MOE 430 2.0 mouse chips. Experiment Overall Design: Number of time points: 2 (6hr and 24 hr) Experiment Overall Design: Number of treatments: 2 (DEX and the vehicle control) Experiment Overall Design: Number of Samples: 3 replicates per time point per treatment Experiment Overall Design: Affymetrix chip: MOE 430 2.0 Experiment Overall Design: Cell type: Primary chondrocyte Experiment Overall Design: Tissue or origin: Tibiae, humerus, femur Experiment Overall Design: Species E15.5 mice Experiment Overall Design: Samples: Total RNA

Project description:Background: Glucocorticoids (GCs) are widely used anti-inflammatory drugs. While useful in clinical practice, patients taking GCs often suffer from skeletal side effects including growth retardation and decreased bone quality in adults. On a physiological level, GCs have been implicated in the regulation of chondrogenesis and osteoblast differentiation, as well as maintaining homeostasis in cartilage and bone. We identified the glucocorticoid receptor (GR) as a potential regulator of chondrocyte hypertrophy in a microarray screen of primary limb bud mesenchyme micromass cultures. Some targets of GC regulation in chondrogenesis are known, but the global effects of pharmacological GC doses on chondrocyte gene expression have not been comprehensively evaluated. Results: This study systematically identifies a spectrum of GC target genes in embryonic growth plate chondrocytes treated with synthetic GR agonist, dexamethasone (DEX), at 6 and 24 hrs. Conventional analysis of this data set and gene set enrichment analysis (GSEA) was performed. Transcripts associated with metabolism were enriched in the DEX condition along with extracellular matrix genes. In contrast, a subset of growth factors and cytokines were negatively correlated with DEX treatment. Comparing DEX-induced gene expression data to developmental changes in gene expression in micromass cultures revealed an additional layer of complexity in which DEX maintains the expression of certain chondrocyte marker genes while inhibiting factors that promote vascularization and ultimately ossification of the cartilaginous template. Conclusions: Together, these results provide insight into the mechanisms and major molecular classes functioning downstream of DEX in primary chondrocytes monolayers. In addition, comparison of our data with microarray studies of DEX treatment in other cell types demonstrated that the majority of DEX effects are tissue-specific. This study provides novel insights into the effects of pharmacological GC on chondrocyte gene transcription and establishes the basis for subsequent functional studies. Keywords: Time course and pharmacological treatment