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Early insights into the role of Exoc6B associated with spondyloepimetaphyseal dysplasia with joint laxity type 3 in primary ciliogenesis and chondrogenic differentiation in vitro

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Abstract

Background

Spondyloepimetaphyseal dysplasia with joint laxity type 3 (SEMDJL3) is a rare skeletal dysplasia associated with EXOC6B, a component of the exocyst complex, involved in vesicle tethering and exocytosis at the plasma membrane. So far, EXOC6B and the pathomechanisms underlying SEMDJL3 remain obscure.

Methods and results

Exoc6b was detected largely at the perinuclear regions and the primary cilia base in ATDC5 prechondrocytes. Its shRNA lentiviral knockdown impeded primary ciliogenesis. In Exoc6b silenced prechondrocytes, Hedgehog signaling was attenuated, including when stimulated with Smoothened agonist. Exoc6b knockdown deregulated the mRNA and protein levels of Col2a1, a marker of chondrocyte proliferation at 7- and 14-days following differentiation. It led to the upregulation of Ihh another marker of proliferative chondrocytes. The levels of Col10a1, a marker of chondrocyte hypertrophy was enhanced at 14 days of differentiation. Congruently, Axin2, a canonical Wnt pathway modulator that inhibits chondrocyte hypertrophy was repressed. The expression of Mmp13 and Adamts4 that are terminal chondrocyte hypertrophy markers involved in extracellular matrix (ECM) remodelling were downregulated at 7 and 14 days of chondrogenesis. Bglap that encodes for the most abundant non-collagenous bone matrix constituent and promotes ECM calcification was suppressed at 14 days of chondrocyte differentiation. ECM mineralization was assessed by Alizarin Red staining. Gene expression and ciliogenesis were investigated by reverse transcription quantitative real-time PCR, immunoblotting, and immunocytochemistry.

Conclusions

These findings provide initial insights into the potential role of Exoc6b in primary ciliogenesis and chondrogenic differentiation, contributing towards a preliminary understanding of the molecular pathomechanisms underlying SEMDJL3.

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Data availability

The data presented in this study are available upon request from the corresponding author.

Abbreviations

SEMDJL3:

Spondyloepimetaphyseal dysplasia with joint laxity, type 3

MTC:

Multisubunit tethering complexes

SNARE:

N-Ethylmaleimide-sensitive factor attachment protein receptors

GTPases:

Guanosine triphosphatases

CATCHR:

Complexes associated with tethering containing helical rods

ERK1/2:

Extracellular signal-regulated kinase 1 and 2

PI(4,5)P2 :

Phosphatidylinositol 4,5 biphosphate

IFT:

Intraflagellar transport

KD:

Knockdown

SAG:

Smoothened agonist

Col2a1:

Type II collagen alpha 1

Hh:

Hedgehog

Ihh:

Indian hedgehog

Col10a1:

Type X collagen alpha 1

Mmp13:

Matrix metallopeptidase 13

Adamts4:

ADAM metallopeptidase with thrombospondin type 1 Motif 4

Bglap:

Bone-gamma carboxyglutamate protein

DMEM:

Dulbecco’s modified eagle medium

FBS:

Fetal bovine serum

PFA:

Paraformaldehyde

DAPI:

4′,6-Diamidino-2-phenylindole

PBS:

Phosphate-buffered saline

shRNA:

Short-hairpin RNA

PCR:

Polymerase chain reaction

RT-PCR:

Reverse transcription PCR

RT-qPCR:

Reverse transcription quantitative real time PCR

SDS-PAGE:

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis

RT:

Room temperature

SEM:

Standard error of the mean

ANOVA:

Analysis of variance

ns:

Non-significant

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Acknowledgements

We are grateful to Dr. Uwe Kornak, Charité—Universitätsmedizin Berlin for ATDC5 cells.

Funding

This work was supported by an Early Career Research award (ECR/2016/001475) for the project entitled ‘Investigating the role of Ift52 and Exoc6B in human biology and disease using cell culture and Drosophila systems’ from the Science and Engineering Research Board, Department of Science and Technology, Government of India and the project entitled ‘Investigating the crosstalk between primary cilia and autophagy in chondrogenesis and its modulation by Fibroblast growth factor (FGF) signaling in FGFR3 related skeletal dysplasias in vitro’ (2020 -107/CMB/ADHOC-BMS) funded by the Indian Council for Medical Research, Government of India to Priyanka Upadhyai.

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Author notes

  1. Vishal Singh Guleria and Neha Quadri have contributed equally to this work.

Authors and Affiliations

  1. Department of Medical Genetics, Kasturba Medical College, Manipal, Manipal Academy of Higher Education, Manipal, India

    Vishal Singh Guleria, Neha Quadri & Priyanka Upadhyai

  2. Department of Cell and Molecular Biology, Manipal School of Life Sciences, Manipal Academy of Higher Education, Manipal, India

    Keshava Prasad

  3. Division of Data Analytics, Bioinformatics and Structural Biology, Yenepoya Research Centre, Yenepoya (Deemed to be University), Mangalore, India

    Ranajit Das

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  1. Vishal Singh Guleria
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Contributions

PU: Conceptulization, Methodology, Resources, Validation, Formal Analysis, Visualization, Writing-original draft, Writing-review and editing, Supervision, Funding acquisition, Project administration. VSG: Methodology, Investigation, Validation, Visualization, Data curation, Formal Analysis, Writing-original draft. NQ: Methodology, Investigation, Validation, Visualization, Data curation, Formal Analysis, Writing-review and editing. KP: Investigation, Validation, Visualization. RD: Formal Analysis, Validation, Visualization, Writing-review and editing. All authors have read and agreed to the submitted version of the manuscript.

Corresponding author

Correspondence to Priyanka Upadhyai.

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Guleria, V.S., Quadri, N., Prasad, K. et al. Early insights into the role of Exoc6B associated with spondyloepimetaphyseal dysplasia with joint laxity type 3 in primary ciliogenesis and chondrogenic differentiation in vitro. Mol Biol Rep 51, 274 (2024). https://doi.org/10.1007/s11033-023-09114-9

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