Zinc Homeostasis in Bone: Zinc Transporters and Bone Diseases

Zinc is an essential micronutrient that plays critical roles in numerous physiological processes, including bone homeostasis. The majority of zinc in the human body is stored in bone. Zinc is not only a component of bone but also an essential cofactor of many proteins involved in microstructural stability and bone remodeling. There are two types of membrane zinc transporter proteins identified in mammals: the Zrt- and Irt-like protein (ZIP) family and the zinc transporter (ZnT) family. They regulate the influx and efflux of zinc, accounting for the transport of zinc through cellular and intracellular membranes to maintain zinc homeostasis in the cytoplasm and in intracellular compartments, respectively. Abnormal function of certain zinc transporters is associated with an imbalance of bone homeostasis, which may contribute to human bone diseases. Here, we summarize the regulatory roles of zinc transporters in different cell types and the mechanisms underlying related pathological changes involved in bone diseases. We also present perspectives for further studies on bone homeostasis-regulating zinc transporters.


Introduction
Bone is the main supporting framework of the body and is characterized by its nature of rigidity and self-healing ability [1]. It provides protection for soft organs and a stable environment for the marrow, which is responsible for hemopoiesis and fat storage [2,3]. Bone is a highly dynamic tissue that undergoes constant physiological activities to remove old, injured or microdamaged bone and replace it with newly formed bone that is mechanically stronger in promoting or preserving bone strength and adapting to changing biomechanical forces [4]. Healthy bones are always in homeostasis, and disturbance of homeostasis causes various bone diseases, such as osteoporosis and osteoarthritis (OA) [5][6][7]. Bone homeostasis involves multiple coordinated cellular and molecular events that revolve around three main types of bone cells: osteoblasts, osteocytes and osteoclasts [8]. Osteoblasts are large cuboidal cells that lie along the bone surface and are responsible for osteogenesis, contributing to bone matrix synthesis and subsequent mineralization [9]. Osteoblasts produce and secrete cellular products during bone formation, are trapped in lacunae and eventually transform into osteocytes [10]. Osteocytes are the most abundant cell type in bone, and they serve as the primary mechanosensor and respond to hormone cues [11,12]. Osteoclasts are large multinucleated cells that are responsible for osteoclastogenesis, accounting for bone destruction and resorption [13]. Osteoblasts originate from mesenchymal stem/stromal cells, whereas osteoclasts are derived from monocyte fusion and have Table 1. Zrt-and Irt-like protein (ZIP) and zinc transporter (ZnT) proteins in bone physiology and pathology.

ZIP1, ZIP2 and ZIP3
Zrt/Irt-like proteins 1, 2 and 3 (ZIP1, ZIP2, and ZIP3) are multipass membrane zinc transporter proteins that are responsible for mammalian zinc homeostasis by mediating zinc uptake into the cytoplasm [58,59]. Osteoblasts are known to be the primary functional cells for bone formation. ZIP1 was found to be expressed early in mouse embryo development, at E15.5 in osteoblasts and later in ameloblasts and odontoblasts, indicating that ZIP1 is closely related to bone formation [60]. Additionally, ZIP1 is located in the cytoplasmic membrane and mediates zinc influx during mesenchymal stem cell (MSC) differentiation into osteoblasts [40]. ZIP1 expression was induced upon osteogenic differentiation of multipotent human MSCs and osteoblast progenitor MC3T3-E1 cells in vitro, accompanied by an increase in intracellular zinc uptake [39][40][41]. Moreover, ZIP1-mediated zinc influx gave rise to higher expression of the osteogenic master regulators Runx2 and Osterix. Enhancement of Runx2 and Osterix expression further modulated transcriptional expression of ZIP1 by directly binding to the response elements in the promoter. These processes resulted in a series of intertwined feed-forward loops that induced zinc influx and osteogenic differentiation and promoted bone apatite formation [41,61,62]. On the other hand, ZIP1 is also expressed in osteoclasts, and overexpression of ZIP1 inhibited mature osteoclast activity by exerting a negative impact on the nuclear factor kappa B (NF-κB) pathway, which is essential for osteoclast differentiation and activity [63]. Thus, ZIP1 could play regulatory roles in stimulating osteogenesis and suppressing osteoclastogenesis in vitro. Although ZIP1 is widely expressed in mouse tissues, no visible bone phenotype was observed in ZIP1 knockout mice fed a normal diet; however, a decreased embryonic survival rate was found upon feeding with a low-zinc diet, but no obvious bone phenotype was revealed [42][43][44].
Unlike that of ZIP1, the expression of ZIP2 and ZIP3 is low in tissues, and few studies have been published on the roles of ZIP2 and ZIP3 in osteogenesis or osteoclastogenesis in vitro. Although ZIP2 expression has been reported to correlate with hormones, no obvious bone phenotypes appeared in homozygous ZIP2 knockout mice with an adequate dietary zinc supply [43][44][45]. Similarly, ZIP3 knockout mice; double ZIP1 and ZIP3 knockout mice; and even triple ZIP1, ZIP2, and ZIP3 knockout mice exhibited no differences in the content of bone zinc and no bone deformity, but the triple knockout mice exhibited a higher risk of malformations under the condition of dietary zinc deficiency [42][43][44]. These interesting phenomena imply that there might be other compensatory pathways that contribute to the process of zinc uptake in vivo.

ZIP8
Zrt/Irt-like protein 8 (ZIP8) is an 8-transmembrane transporter that is present on the cytoplasmic membrane or intracellular vesicles of various types of cells [64,65]. Zinc supplementation or overexpression of ZIP8 in osteoblasts could improve zinc homeostasis, which helps to protect against intermittent hypoxia (IH) exposure in vitro [66]. IH-treated osteoblasts exhibited reductions in mineralization and zinc content, accompanied by decreased expression of ZIP8, Runx2, Col1α1, and other osteogenesis-associated genes, which could be recovered by zinc supplementation or ZIP8 overexpression [66]. Notably, IH-treated animals displayed significantly impaired insulin production and bone fragility due to an imbalance of zinc homeostasis [67,68].
Recently, ZIP8-mediated zinc influx has been revealed to be essential for the pathogenesis of osteoarthritis [46]. OA is characterized by cartilage degradation, synovitis, osteophyte formation, and sclerosis of subchondral bone. Tissue destruction is caused by proteins named matrix-degrading enzymes, which are produced by cartilage cells. Matrix-degrading enzymes need zinc to function, implying that zinc levels play an important role in OA [69]. ZIP8 expression was found to be increased in chondrocytes from OA patients or OA mouse models, resulting in increased intracellular zinc concentrations [46,47,70]. ZIP8 is embedded in the plasma membrane of cartilage cells and is involved in transporting zinc into these cells from the outside environment. Mechanistically, zinc influx through ZIP8 activates the nuclear localization of a transcription-activating protein, metal-regulatory transcription factor-1 (MTF1), which in turn upregulates the expression of zinc-dependent metalloprotease matrix-degrading enzymes (MMP3, MMP9, MMP12, MMP13) and ADAM metallopeptidase with thrombospondin type 1 motif 5 (ADAMTS5), induces the destruction of the cartilage extracellular matrix and exacerbates the pathogenesis of OA [46,48,71]. Moreover, overexpression or transgenic manipulation of ZIP8 or MTF1 in chondrocytes causes OA pathogenesis in mice. It is worth noting that the pathogenesis of OA caused by overexpression of ZIP8 was significantly inhibited in MTF1-deficient mice, whereas the pathogenesis of OA caused by overexpression of MTF1 was not affected in ZIP8-deficient mice. This clearly shows that MTF1 is a downstream mediator of ZIP8 in the pathogenesis of OA. In contrast, chondrocyte-specific deficiency of ZIP8 or MTF1 ameliorated cartilage destruction in OA mice [46]. Furthermore, the zinc-ZIP8-MTF1 axis could reciprocally activate hypoxia-inducible factor 2α (HIF-2α), one of the key regulatory factors of endochondral osteogenesis and OA-related cartilage degeneration, to amplifying catabolic signals and accelerating cartilage destruction [49,72,73]. Therefore, the zinc-ZIP8-MTF1 axis might be a novel drug target for the treatment of OA.

ZIP13
Zrt-and Irt-like protein 13 (ZIP13) possesses an 8-transmembrane structure and acts as a zinc-influx transporter, moving zinc from the subcellular compartment into the cytosol [74]. ZIP13 is located in the Golgi apparatus and intracellular vesicles and mainly plays roles in mesenchyme-originating cells [51,[74][75][76]. ZIP13 can respond to and be regulated by zinc concentrations. This process might contribute to the influx of stored zinc from vesicles to the cytosol, meeting the requirement for zinc in order to activate enzymes and related metabolic reactions under zinc-restricted conditions [76,77]. ZIP13 was found to be expressed in osteoblasts of the tibia and alveolar bone, in the proliferative zone of the growth plate, in odontoblasts of the forming dentin crown in molars, and in fibroblasts of the reticular layer of the skin [51,78,79].
The spondylocheiro dysplastic form of Ehlers-Danlos syndrome (SCD-EDS) is an inherited connective tissue and bone disease caused by a homozygous mutation of the ZIP13 gene [50,77]. Zinc deficiency can cause bone growth retardation and increase the risk of skin fragility [80,81]. Many clinical features of SCD-EDS patients are similar to symptoms of zinc deficiency in humans or animal models, such as osteogenesis imperfecta, skin inelasticity, and systemic growth retardation, which is also recapitulated in ZIP13-knockout mice [50,51]. Mice with ZIP13 deletion exhibited impaired bone morphogenetic protein 4 (BMP4)/transforming growth factor β (TGF-β)/Smad signaling in the corresponding tissues. Bone morphogenetic proteins and TGF-β signaling are known to play important roles in cell proliferation and differentiation and are highly correlated with the development of bone and various tissues [82]. Ablation of ZIP13 initiated dysregulation of a variety of differentiation-related marker genes leading to inadequate maturation of osteoblasts, chondrocytes, odontoblasts and fibroblasts; these markers included 1) Runx2 and Msh homeobox 2 (Msx2), which are involved in skeletogenesis; 2) Indian hedgehog (Ihh), Fibroblast growth factor receptor 3 (Fgfr3) and SRY-box transcription factor 9 (Sox9), which are responsible for differentiation of chondrocytes; and 3) Collagen type I alpha 2 (Col1α2), which is responsible for fibroblast differentiation [51,75]. Furthermore, the valosin-containing protein (VCP)-dependent ubiquitin proteasome pathway accelerates the degradation of the pathological mutant ZIP13 protein, reduces the level of functional protein, and leads to vertebral dysplasia in SCD-EDS [78,83].

ZIP14
ZIP14 is most closely related to ZIP8, which is known to be localized to the plasma membrane and to function in the transport of Zn 2+ , Fe 2+ , and Mn 2+ [84][85][86]. ZIP14 is important for mammalian development and was found to be highly expressed in the pituitary gland and bone, which are responsible for the production of growth hormone and bone elongation [52,53]. ZIP14 is highly expressed in chondrocytes in the proliferation zone of growth plates and contributes to endochondral ossification, which accounts for longitudinal lengthening of bones. Loss of ZIP14 in mice results in dwarfism, scoliosis, osteopenia and shortening of the long bones caused by abnormal chondrogenesis and endochondral ossification [52,53]. At the same time, intracellular zinc concentrations in the growth plate proliferation zone as well as zinc concentrations and 3 ,5 -Cyclic adenosine monophosphate (cAMP) levels in pituitary cells of ZIP14 knockout mice were significantly reduced, and these effects were important for maintaining the homeostasis of G protein-coupled receptor (GPCR)-mediated signal transduction. Further research on the underlying mechanisms revealed that zinc transport mediated by ZIP14 could prevent cAMP degradation by inhibiting phosphodiesterase (PDE) activity, through which ZIP14 facilitates the GPCR/cAMP/cAMP response element-binding protein (CREB) signaling pathways [52,53]. The expression of ZIP14 is stable during osteogenic differentiation of mouse MSCs, whereas it increases during the mineralization stage. ZIP14 knockout mice exhibited markedly reduced trabecular bone mass that was greatly amplified with aging [87]. In humans, hyperostosis cranialis interna (HCI) is a genetic disease related to a missense mutation (P.L441R) in the ZIP14 gene, which was confirmed by whole-exome sequencing [88]. P.L441R-ZIP14 leads to incorrect localization of ZIP14 in osteoblasts, thus increasing the accumulation of unstable cellular zinc and abnormal cellular zinc homeostasis, causing excessive bone growth in the skull [54]. Notably, ZIP14L438R osteoblast-specific knock-in mice, which have a missense mutation in the mouse ZIP14 gene, exhibit no effects on the calvariae but have a severe femoral phenotype characterized by a sharp increase in cortical thickness, similar to the underlying pathology of HCI patients. Conditional osteoblast ZIP14-mutation knock-in mice have a significant increase in cortical thickness and a decreased midshaft due to enhanced intimal formation caused by significantly enhanced cAMP/CREB and nuclear factor of activated T cells (NFAT) signaling activity, which plays an important role in bone and inflammatory processes; however, conditional osteoclast ZIP14-mutation knock-in mice display no significant difference in skeletal phenotype.

Role of ZnT Transporters in Bone
The zinc transporter (ZnT)/Slc30 family is a subfamily responsible for zinc efflux, in contrast to the function of the (ZIP)/Slc39 subfamily [89]. Few studies have been reported on the relationship between the ZnT family and bone, except for ZnT5 and ZnT7. ZnT5 expressed after transfection in HeLa cells was reported to localize to Golgi membranes, mediating zinc efflux from the cytosol to the Golgi compartment [90]. ZnT7 is localized to the Golgi apparatus and unique vesicular compartments [91]. The activity of tissue-nonspecific alkaline phosphatase (TNAP) was significantly reduced in DT40 cells in the absence of both ZnT5 and ZnT7 [92,93]. As a zinc-requiring membrane-bound enzyme, TNAP is crucial for bone mineralization. Decreased TNAP activity inevitably leads to impaired mineralization of the bone matrix, which mimics the skeletal defects of infantile hypophosphatasia [94]. Mice with ZnT5 deficiency displayed poor growth, osteopenia and heart failure [55]. Specifically, ZnT5 knockout mice manifest bone abnormalities, including lower bone density due to impairment of osteoblast maturation [55]. However, the roles of ZnT5 in bone and the related mechanisms are largely elusive. The ZnT7 protein is abundantly expressed in mouse osteoblast MC3T3-E1 cells [57] and is a crucial component of the functional activities of MSCs [56]. ZnT7 prevented MC3T3-E1 cells from undergoing apoptosis induced by oxidative stress by reducing the accumulation of intracellular cationic zinc [57]. On the other hand, the expression of ZnT7 decreased upon osteogenic induction of MSCs. Downregulation of ZnT7 expression by small interfering RNA (siRNA) increased the levels of osteogenesis-associated markers such as alkaline phosphatase (ALP), collagen I and osteocalcin, whereas overexpression of ZnT7 exerted the opposite effects. Further study demonstrated that ZnT7 could interfere with Wnt/β-catenin signaling, a key pathway involved in osteogenesis [56]. ZnT5 and ZnT7 might play opposite roles in osteogenesis; however, the underlying mechanism still needs to be explored.

Conclusions and Future Prospects
Zinc is an important element for bone development, regeneration, and homeostasis, which has been gradually recognized in recent years [95]. However, zinc is incapable of passing through the cell membrane freely, and transporter-mediated influx and efflux of zinc are required for zinc homeostasis in cells. Since the discovery of zinc transporters, accumulating evidence has demonstrated that mutation and disorder of ZIP and ZnT transporter proteins are related to certain aspects of bone physiology or disease [96]. In this review, eight zinc transporters, namely, ZIP1, ZIP2, ZIP3, ZIP8, ZIP13, ZIP14, ZnT5, and ZnT7, were characterized to illuminate the relationship between zinc transporters and bone homeostasis (Figure 1). Different members of the ZIP and ZnT families synergize with zinc to participate in the processes of osteogenesis and osteoclastogenesis. The ZIP8-mediated zinc signaling axis in chondrocytes is an essential regulator of OA pathogenesis; dysfunction of the ZIP13, ZIP14, or ZnT5 gene gives rise to bone deformity in mice and humans. Mutations in zinc transporter genes may lead to genetic disease or increase the risk of disease. However, studies related to the role of zinc transporters in the pathogenesis of bone diseases are relatively rare, and further investigation is needed to gain a deeper understanding, which might facilitate the development of zinc supplements for bone diseases, zinc-containing bone repair materials, and molecular therapies targeting zinc signaling pathways. In addition, recent studies suggest that zinc transporters can transport not only zinc but also trace elements such as iron, manganese, and cadmium, which makes the functions of zinc transporters in bone more complex and diverse. Whether a specific zinc transporter transports other divalent metal ions, whether the same zinc transporter regulates bone development by coordinating the steady-state metabolism of different trace elements, and whether the transport function of a zinc transporter for multiple trace elements is independent or balanced are important scientific issues to be further studied.
suggest that zinc transporters can transport not only zinc but also trace elements such as iron, manganese, and cadmium, which makes the functions of zinc transporters in bone more complex and diverse. Whether a specific zinc transporter transports other divalent metal ions, whether the same zinc transporter regulates bone development by coordinating the steady-state metabolism of different trace elements, and whether the transport function of a zinc transporter for multiple trace elements is independent or balanced are important scientific issues to be further studied.

Conflicts of Interest:
The authors declare no conflict of interest.