MMP14 cleaves PTH1R in the chondrocyte derived osteoblast lineage, curbing signaling intensity for proper bone anabolism

Bone homeostasis is regulated by hormones such as parathyroid hormone (PTH). While PTH can stimulate osteo-progenitor expansion and bone synthesis, how the PTH-signaling intensity in progenitors is controlled is unclear. Endochondral bone osteoblasts arise from perichondrium-derived osteoprogenitors and hypertrophic chondrocytes (HC). We found, via single-cell transcriptomics, HC descendent cells activate membrane-type 1 metalloproteinase 14 (MMP14) and the PTH pathway as they transition to osteoblasts in neonatal and adult mice. Unlike Mmp14 global knockouts, postnatal day 10 (p10) HC lineage-specific Mmp14 null mutants (Mmp14deltaHC) produce more bone. Mechanistically, MMP14 cleaves the extracellular domain of PTH1R, dampening PTH signaling, and consistent with the implied regulatory role, in Mmp14deltaHC mutants, PTH signaling is enhanced. We found HC-derived osteoblasts contribute ~50% of osteogenesis promoted by treatment with PTH 1-34 and this response was amplified in Mmp14deltaHC. MMP14 control of PTH signaling likely applies also to both HC- and non-HC-derived osteoblasts because their transcriptomes are highly similar. Our study identifies a novel paradigm of MMP14 activity-mediated modulation of PTH signaling in the osteoblast lineage, contributing new insights into bone metabolism with therapeutic significance for bone-wasting diseases. We performed single-cell transcriptomics on mouse tibia at week 8 (P56) post natal, with cells pooled from three mice. All the mice had the genotype of MMP14 F/+; COL10A1cre; R26-tdT; Col1a1-2.3-GFP. The raw data was aligned to mouse genome mm10, and a reference of exogenenous RNAs, including GFP, tdTomato, and WPRE.骨稳态受甲状旁腺激素（parathyroid hormone, PTH）等激素调控。尽管PTH可促进骨祖细胞扩增与骨合成，但目前尚不清楚祖细胞内PTH信号的强度是如何被调控的。软骨内骨的成骨细胞来源于软骨膜源性骨祖细胞与肥大软骨细胞（hypertrophic chondrocytes, HC）。本研究通过单细胞转录组学发现，在新生及成年小鼠体内，HC的子代细胞在向成骨细胞转化的过程中，会激活膜型1金属蛋白酶14（membrane-type 1 metalloproteinase 14, MMP14）与PTH信号通路。与Mmp14全身敲除小鼠不同，出生后第10天（postnatal day 10, p10）的HC谱系特异性Mmp14敲除突变体（Mmp14deltaHC）可产生更多骨量。从机制上来说，MMP14可切割甲状旁腺激素1受体（parathyroid hormone 1 receptor, PTH1R）的胞外结构域，从而抑制PTH信号通路；这与我们推测的调控作用一致，在Mmp14deltaHC突变体中，PTH信号通路被增强。我们发现，PTH 1-34治疗所促进的骨生成中，约有50%来自HC源性成骨细胞，且该效应在Mmp14deltaHC突变体中被进一步放大。MMP14对PTH信号通路的调控作用可能同样适用于HC源性与非HC源性成骨细胞，因为二者的转录组特征高度相似。本研究揭示了一种全新的范式：即MMP14通过其活性调控成骨细胞谱系内的PTH信号通路，为骨代谢研究提供了新视角，同时对骨丢失类疾病的治疗具有重要参考价值。我们对出生后第8周（postnatal day 56, P56）的小鼠胫骨进行了单细胞转录组测序，所用细胞来自3只小鼠的混合样本。所有小鼠的基因型均为MMP14 F/+; COL10A1cre; R26-tdT; Col1a1-2.3-GFP。原始测序数据比对至小鼠基因组mm10以及包含绿色荧光蛋白（green fluorescent protein, GFP）、tdTomato与土拨鼠肝炎病毒转录后调控元件（woodchuck hepatitis virus posttranscriptional regulatory element, WPRE）的外源性RNA参考序列集。