Effects of DSPP and MMP20 Silencing on Adhesion, Metastasis, Angiogenesis, and Epithelial-Mesenchymal Transition Proteins in Oral Squamous Cell Carcinoma Cells.

Recent reports highlight the potential tumorigenic role of Dentin Sialophosphoprotein (DSPP) and its cognate partner Matrix Metalloproteinase 20 (MMP-20) in Oral Squamous Cell Carcinomas (OSCCs). However, the function/mechanism of these roles is yet to be fully established. The present study aimed to investigate the effects of DSPP and MMP20 silencing on specific proteins involved in oral cancer cell adhesion, angiogenesis, metastasis, and epithelial-mesenchymal transition (EMT). Stable lines of DSPP/MMP20 silenced OSCC cell line (OSC2), previously established via lentiviral-mediated shRNA transduction, were analyzed for the effects of DSPP, MMP20, and combined DSPP-MMP20 silencing on MMP2, MMP9, integrins αvß3 and αvß6, VEGF, Kallikerin- 4,-5,-8,-10, E-cadherin, N-cadherin, Vimentin, met, src, snail, and Twist by Western blot. Results show a significant decrease (p < 0.05) in the expression of MMP2, MMP9, integrin αvß3, αvß6, VEGF, Kallikerins -4, -5, -8, -10, N-cadherin, vimentin met, src, snail and twist following DSPP and MMP20 silencing, individually and in combination. On the other hand, the expression of E-cadherin was found to be significantly increased (p < 0.05). These results suggest that the tumorigenic effect of DSPP and MMP20 on OSC2 cells is mediated via the upregulation of the genes involved in invasion, metastasis, angiogenesis, and epithelial-mesenchymal transition (EMT).

DSPP-MMP20 gene silencing downregulates cancer stem cell markers in human oral cancer cells.

BACKGROUND: Recent findings indicate that dentin sialophosphoprotein (DSPP) and matrix metalloproteinase (MMP) 20 interact in oral squamous cell carcinoma (OSCC). The objective of this study was to determine the effects of DSPP/MMP20 gene silencing on oral cancer stem cell (OCSC) markers. METHODS: The expression of well-established OCSC markers: ABCG2; ALDH1; CD133; CD44; BMI1; LGR4, and Podoplanin in DSPP/MMP20-silenced OSCC cell line, OSC2, and controls were assayed by western blot (WB), and flow cytometry techniques. The sensitivity of OSC2 cells to cisplatin following DSPP/MMP20 silencing was also determined. RESULTS: DSPP/MMP20 silencing resulted in downregulation of OCSC markers, more profoundly ABCG2 (84%) and CD44 (81%), following double silencing. Furthermore, while treatment of parent (pre-silenced) OSC2 cells with cisplatin resulted in upregulation of OCSC markers, DSPP/MMP20-silenced OSC2 cells similarly treated resulted in profound downregulation of OCSC markers (72 to 94% at 50 µM of cisplatin), and a marked reduction in the proportion of ABCG2 and ALDH1 positive cells (~ 1%). CONCLUSIONS: We conclude that the downregulation of OCSC markers may signal a reduction in OCSC population following MMP20/DSPP silencing in OSCC cells, while also increasing their sensitivity to cisplatin. Thus, our findings suggest a potential role for DSPP and MMP20 in sustaining OCSC population in OSCCs, possibly, through mechanism(s) that alter OCSC sensitivity to treatment with chemotherapeutic agents such as cisplatin.

Metastatic renal cell carcinoma to the oral cavity as first sign of disease: A case report.

Renal cell carcinoma metastasis to the oral cavity is rare. Significantly, the oral lesion in this case was the first indication of a malignant disease in the patient. This case underscores the importance of detailed history taking, interpretation of clinical finding, and high index of suspicion for metastatic disease to the oral cavity.

Survey of dentin sialophosphoprotein and its cognate matrix metalloproteinase-20 in human cancers.

BACKGROUND: Matrix metalloproteinases-20 (MMP20) expression is widely regarded as tooth specific, with expression limited to dental hard tissues. Recently, we reported MMP20 expression and interaction with dentin sialophosphoprotein (DSPP), a member of the Small Integrin Binding Ligand N-linked Glycoproteins (SIBLINGs), in human oral squamous cell carcinoma (OSCC) and dysplastic oral premalignant lesions (OPLs), suggesting a role for MMP20-DSPP interaction in oral carcinogenesis. METHODS: This study aimed to survey the expression of MMP20 and its cognate DSPP partner in the breast, colon, prostate, thyroid, and cervical neoplasms. Using commercially available tissue microarrays (TMAs) and cell lines, we performed immunohistochemistry, immunofluorescence, proximity ligation assay, and western blot experiments to determine the expressions of MMP20 and DSPP in the breast, colon, prostate, thyroid, cervical neoplasms, and their normal counterparts. RESULTS: Significantly high expression levels of MMP20 and DSPP were observed in the malignant breast, colon, prostate, thyroid, and cervical neoplasms compared with their benign and normal counterparts. Furthermore, MMP20 levels increased with advanced stages of colon and thyroid cancers. DSPP expression increased significantly with tumor stage in all cancers examined. CONCLUSIONS: The co-localization and potential MMP20-DSPP interaction previously reported in oral cancers are present in other cancers. These results suggest MMP20-DSPP pairing as a potential marker of disease activity in some epithelial cancers with diagnostic and prognostic implications.

Development and Validation of a Combined Hypoxia and Immune Prognostic Classifier for Head and Neck Cancer.

PURPOSE: Intratumoral hypoxia and immunity have been correlated with patient outcome in various tumor settings. However, these factors are not currently considered for treatment selection in head and neck cancer (HNC) due to lack of validated biomarkers. Here we sought to develop a hypoxia-immune classifier with potential application in patient prognostication and prediction of response to targeted therapy. EXPERIMENTAL DESIGN: A 54-gene hypoxia-immune signature was constructed on the basis of literature review. Gene expression was analyzed in silico using the The Cancer Genome Atlas (TCGA) HNC dataset (n = 275) and validated using two independent cohorts (n = 130 and 123). IHC was used to investigate the utility of a simplified protein signature. The spatial distribution of hypoxia and immune markers was examined using multiplex immunofluorescence staining. RESULTS: Unsupervised hierarchical clustering of TCGA dataset (development cohort) identified three patient subgroups with distinct hypoxia-immune phenotypes and survival profiles: hypoxialow/immunehigh, hypoxiahigh/immunelow, and mixed, with 5-year overall survival (OS) rates of 71%, 51%, and 49%, respectively (P = 0.0015). The prognostic relevance of the hypoxia-immune gene signature was replicated in two independent validation cohorts. Only PD-L1 and intratumoral CD3 protein expression were associated with improved OS on multivariate analysis. Hypoxialow/immunehigh and hypoxiahigh/immunelow tumors were overrepresented in "inflamed" and "immune-desert" microenvironmental profiles, respectively. Multiplex staining demonstrated an inverse correlation between CA-IX expression and prevalence of intratumoral CD3+ T cells (r = -0.5464; P = 0.0377), further corroborating the transcription-based classification. CONCLUSIONS: We developed and validated a hypoxia-immune prognostic transcriptional classifier, which may have clinical application to guide the use of hypoxia modification and targeted immunotherapies for the treatment of HNC.

Effects of oral consumption of the green tea polyphenol EGCG in a murine model for human Sjogren's syndrome, an autoimmune disease.

SIGNIFICANCE: Protection of glandular cells from autoimmune-induced damage would be of significant clinical benefit to Sjogren's syndrome (SS) patients. Epigallocatechin-3-gallate (EGCG) possesses anti-apoptotic, anti-inflammatory, and autoantigen-inhibitory properties. AIMS: To investigate if EGCG protects against certain autoimmune-induced pathological changes in the salivary glands of the non-obese diabetic (NOD) mouse model for SS. MAIN METHODS: Animals were provided with either water or water containing 0.2% EGCG. At the age of 8, 16 and 22 weeks, submandibular salivary gland tissue and serum samples were collected for pathological and serological analysis. KEY FINDINGS: Significant lymphocyte infiltration was observed in the salivary glands of the water-fed group at the age of 16 weeks, while the EGCG group showed reduced lymphocyte infiltration. By 22 weeks of age, water-fed animals demonstrated elevated levels of apoptotic activity within the lymphocytic infiltrates, and high levels of serum total anti-nuclear antibody, compared to EGCG-fed animals. Remarkably, proliferating cell nuclear antigen (PCNA) and Ki-67 levels in the salivary glands of water-fed NOD mice were significantly elevated in comparison to BALB/c control mice; in contrast, PCNA and Ki-67 levels in EGCG-fed NOD animals were similar to BALB/c mice. These results indicate that EGCG protects the NOD mouse submandibular glands from autoimmune-induced inflammation, and reduces serum autoantibody levels. Abnormal proliferation, rather than apoptosis, appears to be a characteristic of the NOD mouse gland that is normalized by EGCG. The evidence suggests that EGCG could be useful in delaying or managing SS-like autoimmune disorders.

The tumorigenic role of DSPP and its potential regulation of the unfolded protein response and ER stress in oral cancer cells.

Dentin sialophosphoprotein (DSPP) is upregulated in various human cancers, including head and neck squamous cell carcinoma. Cancer cells are commonly found under constant endoplasmic reticulum (ER) stress and exhibit increased levels of misfolded proteins, due to gene mutations and a stressful microenvironment. The present study examined the effects of DSPP silencing on the regulation of ER stress and the unfolded protein response (UPR) in oral cancer cells. A recently established stable DSPP short hairpin (sh)RNA-silenced OSC2 oral cancer cell line was used. The mRNA expression levels of ER stress-associated proteins, including 78 kDa glucose-regulated protein (GRP78), sarcoplasmic/endoplasmic reticulum calcium ATPase 2b (SERCA2b), inositol 1,4,5-trisphosphate receptor (IP3r), protein kinase R-like endoplasmic reticulum kinase (PERK), serine/threonine-protein kinase/endoribonuclease IRE1 (IRE1), activating transcription factor 6 (ATF6) and matrix metalloproteinase 20 (MMP20), were assessed by reverse transcription-quantitative polymerase chain reaction. The expression levels of apoptosis-related [B­cell lymphoma 2 (Bcl2), Bcl2-associated X protein (Bax) and cytochrome c] and cell proliferation-related [proliferating cell nuclear antigen (PCNA)] proteins were analyzed by western blotting. Cell viability, apoptosis and migration were monitored by MTT assay, Annexin V-fluorescein isothiocyanate flow cytometry and wound-healing assay, respectively. In transiently transfected puromycin­free OSC2 cells, DSPP silencing markedly downregulated the mRNA expression levels of major ER stress regulators, including GRP78, SERCA2b, PERK, IRE1 and ATF6, as well as MMP20. DSPP silencing also resulted in decreased cell viability and migration, and enhanced apoptosis. Furthermore, PCNA and Bcl2 levels were decreased, whereas Bax and cytochrome c protein levels were increased in DSPP-silenced OSC2 cells. Sustained puromycin treatment partially counteracted the effects of DSPP silencing on the mRNA expression levels of ER stress-related proteins and MMP20, and on the migratory capacity of OSC2 cells. However, following puromycin treatment of DSPP-silenced cells, cell viability was further reduced and apoptosis was enhanced. In conclusion, these data provide evidence to suggest that DSPP may be involved in ER stress mechanisms in oral squamous cell carcinoma, since its downregulation in OSC2 cells led to significant alterations in the levels of major ER stress-associated proteins, and subsequent collapse of the UPR system.

Interferon γ suppresses dentin sialophosphoprotein in oral squamous cell carcinoma cells resulting in antitumor effects, via modulation of the endoplasmic reticulum response.

The expression of proinflammatory cytokines in various malignant neoplasms is widely considered to represent the host immune response to tumor development. The role of interferon (IFN)γ in head and neck squamous cell carcinoma, and its association with endoplasmic reticulum (ER) stress pathways, remains a subject of ongoing investigation. Dentin sialophosphoprotein (DSPP), which is a member of the small integrin­binding N­linked glycoproteins family, has been implicated in malignant transformation and invasion of oral squamous cell carcinoma (OSCC). Recent studies have established matrix metalloproteinase (MMP)20 as the cognate MMP partner of DSPP. The present study examined the effects of IFNγ treatment on DSPP and MMP20 expression, ER stress, the unfolded protein response (UPR), and calcium (Ca) homeostasis regulatory mechanisms in OSCC cells. The OSC2 OSCC cell line was treated with IFNγ at specific time­points. At each time­point, the mRNA expression levels of DSPP and MMP20, and those of ER­stress­, UPR­ and Ca homeostasis­associated proteins [78­kDa glucose­regulated protein (GRP78), sarco/endoplasmic reticulum Ca2+­ATPase (SERCA2b), inositol 1,4,5­trisphosphate receptor (IP3r), protein kinase R­like ER kinase (PERK) and inositol­requiring enzyme 1 (IRE1)], were assessed by reverse transcription­quantitative polymerase chain reaction. The protein expression levels of B­cell lymphoma 2 (Bcl­2), Bcl­2­associated X protein (Bax), proliferating cell nuclear antigen (PCNA) and cytochrome c were analyzed by western blotting. Cell viability, apoptosis and migration were evaluated by MTT, Annexin V­fluorescein isothiocyanate flow cytometry and wound­healing assays, respectively. IFNγ treatment significantly downregulated the mRNA expression levels of the major ER stress regulator GRP78 and, to a lesser extent, the UPR­associated molecule IRE1; however, IFNγ had no significant effect on PERK. With regards to ER Ca homeostasis molecules, treatment with IFNγ downregulated the mRNA expression levels of SERCA2b and upregulated those of IP3r. Furthermore, DSPP and MMP20 mRNA expression levels were significantly reduced following IFNγ treatment. Notably, treatment with IFNγ hampered OSC2 migration, reduced cell viability and PCNA protein expression, enhanced apoptosis, downregulated Bcl­2, and upregulated Bax and cytochrome c. Overall, IFNγ inhibited OSCC cell viability and migration, and increased apoptosis, possibly by regulating ER stress and UPR mechanisms. In addition, IFNγ­induced DSPP and MMP20 downregulation may correspond with alteration in ER Ca homeostasis.

SIBLING expression patterns in duct epithelia reflect the degree of metabolic activity.

The SIBLING (Small Integrin-Binding LIgand, N-linked Glycoprotein) family of secreted glycophosphoproteins includes bone sialoprotein (BSP), dentin matrix protein-1 (DMP1), dentin sialophosphoprotein (DSPP), osteopontin (OPN), and matrix extracellular phosphoglycoprotein (MEPE). For many years, they were thought in normal adults to essentially be limited to metabolically active mesenchymal cells that assembled the mineralized matrices of bones and teeth. Over the last decade they have also been upregulated in a variety of tumors. Three of these proteins (BSP, OPN, and DMP1) have been shown to interact with three matrix metalloproteinases (MMP-2, MMP-3, and MMP-9, respectively). Recently, all five SIBLINGs and their MMP partners when known were observed in specific elements of normal ductal epithelia in salivary gland and kidney. We have hypothesized that the SIBLINGs and their MMP partners may be expressed in ductal cells with high metabolic activity. In this paper, we show that all the SIBLINGs (except MEPE) and their MMP partners are expressed in the metabolically active epithelia of human eccrine sweat gland duct but not in the more passive ductal cells of the macaque (monkey) lacrimal gland. It is hypothesized that MEPE expression may be limited to cells involved in active phosphate transport. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Green tea polyphenols reduce autoimmune symptoms in a murine model for human Sjogren's syndrome and protect human salivary acinar cells from TNF-alpha-induced cytotoxicity.

Sjogren's syndrome (SS) is a relatively common autoimmune disorder. A key feature of SS is lymphocytic infiltration of the salivary and lacrimal glands, associated with the destruction of secretory functions of these glands. Current treatment of SS targets the symptoms but is unable to reduce or prevent the damage to the glands. We reported previously that the major green tea polyphenol (GTP) epigallocatechin-3-gallate (EGCG) inhibits autoantigen expression in normal human keratinocytes and immortalized normal human salivary acinar cells (Hsu et al. 2005). However, it is not known whether GTPs have this effect in vivo, if they can reduce lymphocytic infiltration, or protect salivary acinar cells from tumor necrosis factor-alpha (TNF-alpha)-induced cytotoxicity. Here, we demonstrate that in the NOD mouse, a model for human SS, oral administration of green tea extract reduced the serum total autoantibody levels and the autoimmune-induced lymphocytic infiltration of the submandibular glands. Further, we show that EGCG protected normal human salivary acinar cells from TNF-alpha-induced cytotoxicity. This protection was associated with specific phosphorylation of p38 MAPK, and inhibitors of the p38 MAPK pathway blocked the protective effect. In conclusion, GTPs may provide a degree of protection against autoimmune-induced tissue damage in SS, mediated in part through activation of MAPK elements.

Up-regulation of SIBLING proteins and correlation with cognate MMP expression in oral cancer.

Various combinations of the SIBLING family of proteins have been found to be up-regulated in many human cancers and have been linked to different stages of tumor progression, including metastasis. Bone sialoprotein (BSP), osteopontin (OPN) and dentin matrix protein 1 (DMP1) specifically bind and activate MMP-2, MMP-3, and MMP-9, respectively. These proteases have also been shown to play important roles in oral squamous cell carcinoma (OSCC) invasion and metastasis. However, with the exception of OPN, there are no reports on the expression of the family of five SIBLING proteins in OSCC. This study examines the expression patterns of the SIBLING family (and MMP partners when known) in OSCC, correlating expression to outcome variables. Archived paraffin sections of 87 cases of primary OSCC were screened by immunohistochemistry for the SIBLINGs and their MMP partners. Three SIBLINGs (BSP, DSPP, and OPN), were expressed in OSCC, while DMP1 and MEPE expression were never observed. Furthermore, BSP and OPN were always expressed with their known MMP partners, MMP-2 and MMP-3, respectively. Poorly differentiated tumors exhibited reduced or no immunoreactivity for BSP and OPN but increased immunoreactivity for DSPP. Seventy eight (90%) cases were positive for BSP and DSPP, while 79 cases (91%) were positive for OPN. Overall, 91% of the cases were positive for at least one SIBLING. There were no correlations between SIBLING expression and tumor size ("T"; of the Union Internationale Contre le Cancer [UICC]-TNM classification for OSCC), and between SIBLING expression and lymph node spread for the T1/T2 tumors. The levels of DSPP expression for floor of mouth and retromolar region tumors were higher than for tongue tumors. Statistically significant correlations were, however, found between the expression levels of BSP and MMP-2 (p<0.0001), BSP and MMP-3 (p<0.0001), and OPN and MMP-3 (p<0.0024). We conclude that BSP, DSPP, and OPN are highly up-regulated in OSCC. While the production of these SIBLINGs is independent of T, they correlate with oral location of tumor, cognate MMP expression, and for DSPP, the degree of tumor differentiation.

Huge peripheral osteoma of the mandible: a case report and review of the literature.

Osteomas are rare benign tumors of bone commonly occurring in the maxillofacial skeleton. In the course of their slow but steady increase in size, osteomas of the maxillofacial bones remain asymptomatic until they attain sufficient sizes as to cause disfigurement and/or direct interference with the normal function of their anatomic location. Here, we report a case of a huge solitary peripheral osteoma of the lingual posterior mandible in a 50-year-old woman who was otherwise in good health. The patient reported at the dental clinic because the lesion interfered with speech, swallowing, and caused occasional gagging. Histological examination confirmed the clinical impression of a peripheral osteoma. Treatment was by surgical excision and histological examination. The patient remains free of recurrence after 5 years.

Emerging paradigm of virtual-microscopy for histopathology diagnosis: survey of US and Canadian oral pathology trainees.

OBJECTIVES/AIMS: The application of virtual microscopy (VM) to research, pre-doctoral medical and dental educational training, and diagnostic surgical and anatomic pathology is well-documented but its application to the field of oral and maxillofacial pathology has not been explored. This is the first study to evaluate the enthusiasm and readiness of US-/Canada-based oral and maxillofacial pathology (OMFP) residents toward employing VM use over conventional microscopy (CM) for diagnostic purposes. MATERIALS AND METHODS: All 46 current US-/Canada-based OMFP residents were invited to participate in an anonymous electronic survey via 'Survey Monkey' in 2015. The survey comprised sixteen multiple choice questions and two 'free text' questions. RESULTS: 14% of respondents of the 22 (48%) respondents who completed the survey indicated a willingness to substitute CM with VM in <5 years, and 33% within 10 years. 52% reported they would never substitute CM with VM. Approximately 10 and 57% of respondents thought VM will become an acceptable sole diagnostic tool in most centers within 5 and 10 years, respectively. These findings are irrespective of the fact that overall, 90% of respondents reported being familiar with VM use. DISCUSSION: VM technology is unlikely to substitute CM in diagnostic oral and maxillofacial histopathology practice among future OMFP practitioners in the foreseeable future.

Matrix Metalloproteinase 20 Co-expression With Dentin Sialophosphoprotein in Human and Monkey Kidneys.

We recently reported the expression of matrix metalloproteinase 20 (MMP20), hitherto thought to be tooth specific, in the metabolically active ductal epithelial cells of human salivary glands. Furthermore, our report indicated that MMP20 co-expressed and potentially interacts with dentin sialophosphoprotein (DSPP), a member of the small integrin-binding ligand N-linked glycoproteins (SIBLINGs). Our earlier reports have shown the co-expression of three MMPs, MMP2, MMP3, and MMP9, with specific members of the SIBLING family: bone sialoprotein, osteopontin, and dentin matrix protein 1, respectively. This study investigated the expression of MMP20 and verified its co-expression with DSPP in human and monkey kidney sections and human mixed renal cells by IHC, in situ proximity ligation assay, and immunofluorescence. Our results show that MMP20 is expressed in all segments of the human and monkey nephron with marked intensity in the proximal and distal tubules, and was absent in the glomeruli. Furthermore, MMP20 co-expressed with DSPP in the proximal, distal, and collecting tubules, and in mixed renal cells. Consistent with other SIBLING-MMP pairs, the DSPP-MMP20 pair may play a role in the normal turnover of cell surface proteins and/or repair of pericellular matrix proteins of the basement membranes in the metabolically active duct epithelial system of the nephrons.

Expression of the SIBLINGs and their MMP partners in human benign and malignant prostate neoplasms.

The small integrin binding ligands n-linked glycoproteins (SIBLINGs) have emerged as potential diagnostic and prognostic indices, and as key targets, in cancer therapy. Three members of the SIBLING family: bone sialoprotein (BSP); osteopontin (OPN); and dentin matrix protein1 (DMP1), bind and interact with specific matrix metalloproteinases (MMPs): BSP-MMP2; OPN-MMP3; DMP1-MMP9, in biochemical and biologic systems. The other two family members are dentin sialophosphoprotein (DSPP) and matrix extracellular phosphoglycoprotein (MEPE). The specific SIBLING-MMP pairing reported in some cancers have not been reported in prostate neoplasms. In this study, we investigated SIBLING-MMP expression and potential interaction in prostate neoplasms. Chi square analysis of immunohistochemistry results showed significant upregulation of OPN (X2=25.710/p<0.001), BSP (X2=19.546/p<0.001), and DSPP (X2=8.720/p=0.003) in prostate adenocarcinoma (pAdC). MEPE was significantly upregulated in benign prostate hyperplasia (BPH; X2=44.153/p<0.001). There were no significant differences in MMP expression between BPH and pAdC. Western blot analysis showed significantly elevated BSP and DSPP in prostate cancer-derived cells. Immunofluorescence studies confirmed BSP-MMP2, OPN-MMP3, and DMP1-MMP9 coexpression in two cancer-derived cell lines, whereas in situ proximity ligation assays confirmed potential BSP-MMP2, OPN-MMP3, and DMP1-MMP9 interactions in BPH and pAdC. Our reports provide evidence that SIBLING-MMP interaction may play a role in the progression of BPH to pAdC.

Human osteopetroses and the osteoclast V-H+-ATPase enzyme system.

Osteopetroses are a heterogeneous group of human genetic diseases characterized by generalized increase in bone density due either to a decreased osteoclast population, defect in osteoclast function, or both. Current knowledge of the pathogenesis suggests defects that may be either intrinsic to osteoclast-monocyte lineage or extrinsic to the mesenchymal cells that support osteoblast ontogeny and activation. Four clinically distinct forms of human osteopetroses currently recognized are the infantile malignant autosomal recessive form, the intermediate autosomal recessive form, the adult benign autosomal dominant osteopetrosis type I, and the autosomal dominant osteopetrosis type II. Propensity to fracture is high in all types of osteopetrosis, and other characteristic clinical problems include hematologic and metabolic abnormalities, infections of affected bone, and neurologic sequela. Among the infantile malignant clinical forms 50-60% of patients present with defects in the OC116-KDa (also refers to ATP6i, TCIRGI, a3) subunit of the osteoclast vacuolar H+-ATPase (V-H+-ATPase) proton pump. Approaches that have been applied to the treatment of osteopetrosis include those aimed at stimulating host osteoclasts. These approaches however have met with little success, and it would appear that the future for the successful treatment of osteopetrosis lies with bone marrow transplantation. Various animal models mimicking some of the clinical subtypes of osteopetrosis have been generated in efforts to elicit further understanding of the pathogenesis. This review is an update on the various phenotypic presentations of human osteopetroses alongside their known animal models. Further studies on these animal models will not only expand our basic understanding of the molecular mechanisms of osteopetroses, but will also aid our ability to develop therapeutic means of intervention in diseases involving osteopetroses.