miRNA-based regulation in growth plate cartilage: mechanisms, targets, and therapeutic potential

Abstract

MicroRNAs (miRNAs) are critical regulators of the skeleton. In the growth plate, these small non-coding RNAs modulate gene networks that drive key stages of chondrogenesis, including proliferation, differentiation, extracellular matrix synthesis and hypertrophy. These processes are orchestrated through the interaction of pivotal pathways including parathyroid hormone-related protein (PTHrP), Indian hedgehog (IHH), and bone morphogenetic protein (BMP) signaling. This review highlights the miRNA-mRNA target networks essential for chondrocyte differentiation. Many miRNAs are differentially expressed in resting, proliferating and hypertrophic cartilage zones. Moreover, differential enrichment of specific miRNAs in matrix vesicles is also observed, providing means for chondrocytes to influence the function and differentiation of their neighbors by via matrix vesicle protein and RNA cargo. Notably, miR-1 and miR-140 emerge as critical modulators of chondrocyte proliferation and hypertrophy by regulating multiple signaling pathways, many of them downstream from their mutual target Hdac4. Demonstration that a human gain-of-function mutation in miR-140 causes skeletal dysplasia underscores the clinical relevance of understanding miRNA-mediated regulation. Further, miRNAs such as miR-26b have emerged as markers for skeletal disorders such as idiopathic short stature, showcasing the translational relevance of miRNAs in skeletal health. This review also highlights some miRNA-based therapeutic strategies, including innovative delivery systems that could target chondrocytes via cartilage affinity peptides, and potential applications related to treatment of physeal bony bridge formation in growing children. By synthesizing current research, this review offers a nuanced understanding of miRNA functions in growth plate biology and their broader implications for skeletal health. It underscores the translational potential of miRNA-based therapies in addressing skeletal disorders and aims to inspire further investigations in this rapidly evolving field.

Keywords: SOX9; chondrocyte; growth plate; matrix vesicles; miR-140; microRNA; regenerative medicine; skeletal dysplasia.

Figure 1

MicroRNA biogenesis and mechanism of function. In the nucleus, microRNA genes are transcribed primarily by RNA polymerase II (Pol II) into primary microRNAs (pri-miRNAs). These pri-miRNAs are processed by the Microprocessor complex, comprising Drosha ribonuclease III (DROSHA) and DiGeorge syndrome critical region 8 (DGCR8), to produce precursor microRNAs (pre-miRNAs) of approximately 60–70 nucleotides. The pre-miRNAs are exported from the nucleus to the cytoplasm by an Exportin 5 (XPO5) containing complex. In the cytoplasm, the pre-miRNA is further processed by DICER, to generate a mature miRNA duplex. An Argonaute (AGO) family member is a key component of the miRNA-induced silencing complex (RISC), which unwinds the miRNA duplex and selects the miRNA guide strand. The miRNA-RISC complex binds to target messenger RNAs (mRNAs) via sequence complementarity, leading to mRNA degradation and/or translational repression.

Figure 2

MiRNAs are differentially expressed in growth plate zones. Model of growth plate chondrocyte zones with their marker genes, key soluble signaling molecules and gradients of microRNA expression represented by varying colors across each zone. miRNA profiling data from (13) was used to determine the growth plate zone of expression. References for the function of miRNAs in this figure: miR-143-3p (14, 15), miR-26b-3p (16), miR-140 (–20), miR-199a (21, 22), miR-503-5p (13), miR-335-5p (13, 23) miR-374-5p (13), miR-379-5p (13), miR-26a (24), miR-1 (25, 26), miR-322-5p (27, 28).

Figure 3

Regulatory landscape of miR-140 and other miRNAs expressed primarily in resting and proliferating zone chondrocytes. (A) Graphic depicting the dynamic expression of miR-140, miR-143-3p and miR-335-5p in the growth plate zones. (B) miR-140 is under the control of the master chondrogenic transcription factor SOX9. miR-140 directly targets Dnpep (aspartyl aminopeptidase) and Hdac4. HDAC4 suppresses the activity of the transcription factors MEF2C and RUNX2, which drive hypertrophic differentiation; Dnpep indirectly increases chondrocyte hypertrophy via dampening BMP signaling. (C) miR-143-3p targets Bmpr2 and Ihh. By repressing Bmpr2, miR-143-3p dampens BMP signaling, reducing SOX9 activity and cartilage matrix production. IHH promotes chondrocyte proliferation, which could be limited by miR-143-3p expression. Since miR-1307-3p targets BMPR2, it can limit BMP signaling and chondrogenic differentiation. miR-199a targets Smad1, a SMAD that transduces signaling downstream of BMPR activation. (D) miR-335-5p supports chondrogenesis by targeting negative regulators of several pathways important for chondrogenesis. It directly represses RhoA-associated protein kinase (Rock1) and Dishevelled-associated activator of morphogenesis 1 (Daam1), both of which are suppressors of Sox9. Therefore miR-335-5p enhances SOx9 expression and upregulation of chondrogenic markers. miR-335-5p also targets Dickkopf-1 (Dkk1), an inhibitor of Wnt signaling. Therefore, miR-335-5p amplifies Wnt activity, further promoting chondrogenesis. Increased Wnt signaling promotes Mest transcription, which is the host gene for miR-335, boosting the expression of miR-335 in an amplification loop. miR-495, miR-101, and miR-1247 inhibit chondrocyte differentiation by targeting Sox9.

Figure 4

Regulatory landscape of miRNAs expressed in proliferating and hypertrophic zone chondrocytes. (A) Graphic depicting the dynamic expression of miRNAs active in proliferating and hypertrophic zones. (B) PTHrP and Ihh pathways form an important regulatory loop that controls chondrocyte proliferation and differentiation. PTHrP binds to its receptor PTH1R and induces the expression of miR-503-5p, miR-374-5p, miR-379-5p, which in turn promote chondrocyte proliferation and inhibit hypertrophic differentiation. (C) miR-26b targets A-Kinase Anchoring Protein 2 (Akap2), a scaffold protein that anchors protein kinase A (PKA) and other signaling molecules. AKAP2 activity is important for the phosphorylation of ERK1/2, which is necessary for chondrocyte proliferation and ossification. (D) miR-1 directly targets Hdac4 and Ihh. HDAC4 suppresses RUNX2 transcriptional activity, which drives the expression of Col10a1 and promotes hypertrophic differentiation. RUNX2 induces IHH expression. miR-1 targets Ihh and negatively affects chondrocyte hypertrophy. miR-26a targets Col10a1, negatively regulating extracellular matrix composition. (E) SOX9 increases the expression of miR-322-5p, which targets Smad7, a negative regulator of TGFβ signaling, which is needed for terminal differentiation. By targeting Smad7, miR-322 helps promote terminal differentiation of chondrocytes. miR-322-5p also targets GHR (growth hormone receptor), which is expressed in the resting and proliferating zones of the growth plate and mediates the anabolic effects of growth hormone on the growth plate.