Pagel Charles N, Kularathna Pamu K, Sanaei Reza, Young Neil D, Hooper John D, Mackie Eleanor J
Department of Veterinary Biosciences, Melbourne Veterinary School, Faculty of Veterinary and Agricultural Sciences, University of Melbourne, Parkville, Victoria, Australia.
Mater Research Institute, Translational Research Institute, University of Queensland, Woolloongabba, Queensland, Australia.
Prostate. 2022 May;82(6):723-739. doi: 10.1002/pros.24316. Epub 2022 Feb 15.
Metastatic prostate cancer lesions in the skeleton are frequently characterized by excessive formation of bone. Prostate cancer cells secrete factors, including serine proteases, that are capable of influencing the behavior of surrounding cells. Some of these proteases activate protease-activated receptor-2 (PAR ), which is expressed by osteoblasts (bone-forming cells) and precursors of osteoclasts (bone-resorbing cells). The aim of the current study was to investigate a possible role for PAR in regulating the behavior of bone cells exposed to metastatic prostate cancer cells.
The effect of medium conditioned by the PC3, DU145, and MDA-PCa-2b prostate cancer cell lines was investigated in assays of bone cell function using cells isolated from wildtype and PAR -null mice. Osteoclast differentiation was assessed by counting tartrate-resistant acid phosphatase-positive multinucleate cells in bone marrow cultured in osteoclastogenic medium. Osteoblasts were isolated from calvariae of neonatal mice, and BrdU incorporation was used to assess their proliferation. Assays of alkaline phosphatase activity and quantitative PCR analysis of osteoblastic gene expression were used to assess osteoblast differentiation. Responses of osteoblasts to medium conditioned by MDA-PCa-2b cells were analyzed by RNAseq.
Conditioned medium (CM) from all three cell lines inhibited osteoclast differentiation independently of PAR . Media from PC3 and DU145 cells had no effect on assays of osteoblast function. Medium conditioned by MDA-PCa-2b cells stimulated BrdU incorporation in both wildtype and PAR -null osteoblasts but increased alkaline phosphatase activity and Runx2 and Col1a1 expression in wildtype but not PAR -null cells. Functional enrichment analysis of RNAseq data identified enrichment of multiple gene ontology terms associated with lysosomal function in both wildtype and PAR -null cells in response to MDA-PCa-2b-CM. Analysis of individual genes identified osteogenesis-associated genes that were either upregulated by MDA-PCa-2b-CM selectively in wildtype cells or downregulated selectively in PAR -null cells.
Factors secreted by prostate cancer cells influence bone cell behavior through both PAR -dependent and -independent mechanisms. Both PAR -independent suppression of osteoclast differentiation and PAR -dependent stimulation of osteogenesis are likely to determine the nature of prostate cancer metastases in bone.
骨骼中的转移性前列腺癌病灶通常以过度的骨形成为特征。前列腺癌细胞分泌包括丝氨酸蛋白酶在内的多种因子，这些因子能够影响周围细胞的行为。其中一些蛋白酶可激活蛋白酶激活受体-2（PAR），该受体由成骨细胞（骨形成细胞）和破骨细胞（骨吸收细胞）前体表达。本研究的目的是探讨PAR在调节暴露于转移性前列腺癌细胞的骨细胞行为中的可能作用。
使用从野生型和PAR基因敲除小鼠分离的细胞，在骨细胞功能测定中研究PC3、DU145和MDA-PCa-2b前列腺癌细胞系条件培养基的作用。通过对在破骨细胞生成培养基中培养的骨髓中抗酒石酸酸性磷酸酶阳性多核细胞进行计数来评估破骨细胞分化。从新生小鼠颅骨中分离成骨细胞，并用BrdU掺入法评估其增殖。通过碱性磷酸酶活性测定和成骨细胞基因表达定量PCR分析来评估成骨细胞分化。通过RNA测序分析成骨细胞对MDA-PCa-2b细胞条件培养基的反应。
来自所有三种细胞系的条件培养基均能独立于PAR抑制破骨细胞分化。PC3和DU145细胞的培养基对成骨细胞功能测定无影响。MDA-PCa-2b细胞条件培养基可刺激野生型和成骨细胞PAR基因敲除小鼠的成骨细胞中BrdU掺入，但仅增加野生型细胞而非PAR基因敲除细胞中的碱性磷酸酶活性以及Runx2和Col1a1表达。RNA测序数据的功能富集分析表明，在野生型和成骨细胞PAR基因敲除小鼠的细胞中，响应MDA-PCa-2b条件培养基时，多个与溶酶体功能相关的基因本体术语均有富集。对单个基因的分析确定了成骨相关基因，这些基因在野生型细胞中被MDA-PCa-2b条件培养基选择性上调，而在PAR基因敲除细胞中被选择性下调。
前列腺癌细胞分泌的因子通过PAR依赖和非依赖机制影响骨细胞行为。破骨细胞分化的PAR非依赖性抑制和成骨的PAR依赖性刺激可能共同决定了前列腺癌骨转移的性质。
