The Clec14a protein has been found to play a key role in regulating bone formation and density

Novel research uncovers how the Clec14a protein in capillary endothelial cells controls bone growth and maturation, revealing a key mechanism of skeletal development.

Test: The H-type endothelial cell protein Clec14a coordinates osteoblast activity during trabecular bone formation and modeling.. Image source: NTshutterth/Shutterstock.com

A recent study published in Biology of communication investigated the role of the C-type lectin domain containing 14A (Clec14a), an H-type endothelial protein, in coordinating osteoblast activity.

Background

Recently, increasing attention has been paid to the role of bone angiogenesis in the regulation of postnatal skeletal growth and bone remodeling and repair. Technological progress has led to the discovery of modern subtypes of bone capillaries. H-type capillaries have been reported to be busy modulators of osteogenesis.

Multimerin 2 (MMRN2) is an extracellular matrix protein that simultaneously binds CD248 and CLEC14A. Both regulate pathological and embryonic angiogenesis, cell migration and adhesion.

Previously, the authors reported that Cd248 negatively regulates osteoblast mineralization and its removal increases osteoblast-driven mineralization. Additionally, recent reports suggest that Clec14a levels are increased during bone healing.

Study and conclusions

In the present study, researchers assessed the role of Clec14a in bone formation. They first assessed the expression of Clec14a and its ligand, Mmrn2, in mouse long bone and confirmed that both proteins were highly expressed in the metaphysis.

Furthermore, Clec14a was immunolabeled alive using anti-Clec14a antibody. This indicated that Clec14a localizes mainly to the endothelium of H-type vessels in the metaphysis.

Overall, the investigators confirmed that Mmrn2 and Clec14a expression was restricted to endothelial cells (ECs), which was more pronounced in H-vascular ECs. They then assessed the effect of Clec14a deletion on bone vascularization.

They observed a reduction in the percentage of H-type endothelium Clec 14a^-/- two-week-old mice compared to wild type (WT) Clec 14a^+/+ mice. The number of capillaries in both groups increased from postnatal day 4 (P4) to four weeks.

Moreover, vessel density did not differ significantly between the two groups. Immunofluorescence imaging of the tibia in both groups at P4 revealed a highly connected vascular bed with almost similar L-type and H-type capillary coverage.

However, in juvenile mice (four weeks ancient), both groups showed a reduced H-type placenta compared to P4 mice, clearly located in the upper part of the metaphysis.

Additionally, after four weeks, a reduction in H-column length and area covered by the H-column vascular front was observed in the metaphysis in Clec14a-/- mice.

The team then examined the effects Clec 14a deletion in the localization of mature and immature osteoblasts using immunostaining for collagen type 1 (Col1a1) and Osterix (Osx), respectively.

Osx⁺ osteoblasts were detected in high numbers in the endosteum and metaphysis of the tibia, regardless of genotype, with fewer osteoblasts in the diaphysis. Clec 14a^-/- mice had significantly increased numbers of Osx⁺ osteoblasts in the inferior metaphysis at P4 compared to WT mice.

Additionally, the team measured tibial length and body weight in two-, four-, eight-, and 30-week-old WT and Clec 14a^-/- mice. There were no significant differences in body weight at any time point. Bone elongation was most busy between two and four weeks in both genotypes, when the length of the tibia doubled. Of note, after two weeks, the tibiae were shorter in WT mice than in mice Clec 14a^-/- mice.

Additionally, the researchers observed metatarsal explant cultures for a week at embryonic day 14.5 to understand the impact Clec 14a^-/- deletion at an early stage of development.

They noticed significant growth Clec 14a^—/—explant length at all time points. The team then used RNA sequencing of the skull cell isolates to better understand the molecular changes at the transcript level.

In this way, 1223 genes with differential expression between the two genotypes were identified. Gene set enrichment analysis revealed forceful expression of markers enriched in osteoblast proliferation and maturation and endochondral ossification pathways in Clec 14a^-/- samples.

Furthermore, the expression of the osteoblast maturation gene in WT i calvarial osteoblasts was examined Clec 14a^-/- P4 puppies raised without EC.

Alkaline phosphatase transcript and protein levels (Alpl) have been measured in vitro over 21 days. Alpl transcript levels increased in WT i Clec 14a^-/- osteoblasts in response to maturation stimuli.

However, the top Alpl expression was observed much earlier (day 4) in Clec 14a^-/--isolated osteoblasts than isolated WT osteoblasts (on day 12). Freshly insulated passage Clec 14a^-/--isolated osteoblasts showed growth Alpl activity in response to maturation stimuli on days 6 and 8.

Conclusions

Scientists have shown that Clec14a and Mmrn2 expression is restricted to ECs in the bone stroma; both genes are highly expressed in H-type endothelium and are differentially regulated.

The results also indicate dysregulation of apical cell formation and migration Clec 14a^-/- mice and blunting of H-type vessel buds on the growth plate in four-week-old infants Clec 14a^-/- mice.

Overall, the study identified Clec14a as a key EC-specific target protein whose inactivation enhances bone formation.