FAM98 Family Proteins Play Distinct Roles in Osteoclastogenesis and Bone Resorption
Osteoclasts play a pivotal role in skeleton growth/modeling, homeostasis/remodeling, and fracture repair . The aberrant bone resorption due to either increased or diminished osteoclast number and/or activity is detrimental to skeleton health and causes anomalous bone mass and bone erosion in multiple bone diseases, including osteoporosis and osteopetrosis. Thus, unveiling the molecules and pathways regulating osteoclast differentiation and function under physiological and pathological conditions will not only advance our knowledge in osteoclast biology but also identify therapeutic targets for the treatment of developmental and degenerative bone diseases. Osteoclast differentiation is regulated by two cytokines, M-CSF (macrophage colony-stimulating factor) and RANKL (a receptor activator of nuclear factor-kB ligand) . Upon attachment of osteoclasts to bone matrix, the activating signals triggered by M-CSF and RANKL, as well as those transduced through the integrins and the immunoreceptors, act in concert to promote osteoclast actin cytoskeleton organization to form actin rings at the sealing zone that tightly seal the resorptive microenvironment. Subsequently, the protons and hydrolases are released via lysosome secretion to dissolve bone minerals and degrade bone matrix, respectively. Therefore, both cytoskeleton organization and lysosome secretory pathway are essential for osteoclast activation and bone resorption. Of the key molecules regulating osteoclastogenesis and function identified so far, PLEKHM1 is specifically indispensable for lysosome trafficking and secretion and bone resorption in osteoclasts . Mutations in the PLEKHM1 gene cause an intermediate form of osteopetrosis in humans and in incisors absent ( ia / ia) rats . Moreover, both germline- and osteoclast-specific Plekhm1 knockout mice have high bone mass phenotypes resulting from defective bone resorption . PLEKHM1 regulates osteoclast lysosomal trafficking and formation of the ruffled border through its interaction with RAB7, a small GTPase that specifically regulates lysosomal pathways in eukaryotic and mammalian cells [ , , , ]. RAB7 is localized at the ruffled border in bone-resorbing osteoclasts. Depletion of Rab7 expression or disruption of Plekhm1-Rab7 interaction attenuates lysosome secretion and bone resorption in cultured rat and murine osteoclasts in vitro . In addition to Rab7, we have previously identified several Plekhm1-binding proteins in murine osteoclasts including Fam98a (family with sequence similarity 98 member A) that may mediate PLEKHM1’s function in osteoclasts . The FAM98 family proteins have three members in humans and mice (FAM98A, FAM98B, and FAM98C). Their physiological functions remain largely unknown. FAM98A has been reported as a substrate of protein arginine methyltransferase 1 (PRMT1) and may be involved in tumor cell migration and invasion . It has been found to localize at stress granules upon various stress stimuli . We have unveiled that Fam98a interacts with Plekhm1 in murine osteoclasts and knockdown of Fam98a expression by lentiviral transduction of specific short hairpin RNAs (shRNAs) disrupting lysosome trafficking/secretion and bone resorption in osteoclasts in vitro . FAM98B has been identified as a component of tRNA ligase and RNA capping/transporting complexes regulating RNA metabolism and translation [ , , , ]. FAM98C has been suggested as a candidate gene regulating ciliogenesis, and the mutation of which may cause ciliopathies . The co-expressions and interactions among FAM98A/B/C in mammalian cells and tissues have not been reported. In this study, we investigated the roles of Fam98 family proteins in osteoclastogenesis and bone resorption by the generation of Fam98a myeloid conditional knockout mice and the depletion of Fam98b and Fam98c expression by lentiviral transduction of specific shRNAs in control and Fam98a-deficient bone marrow monocytes. 

2.1. Reagents The reagents used in this study were listed in . 2.2. Bone Marrow Monocyte and Osteoclast Cultures Whole bone marrow cells were extracted from the tibias and femurs of 10-week-old mice. Bone marrow monocytes (BMMs) and osteoclasts were cultured as described previously. In brief, red blood cells were lysed in 1× red blood cell lysis buffer for 5 min at room temperature. Bone marrow cells were plated onto a 100 mm Petri dish and cultured in α-MEM containing 10% heat-inactivated FBS, 1× Penicillin–Streptomycin-L-Glutamine (PSG), and 1/10 volume of CMG 14–12 (conditioned medium supernatant containing recombinant M-CSF at 1 μg/mL)  for 4 days. To generate osteoclasts from BMMs, 1.5 × 10 4 or 3 × 10 4 BMMs were cultured in α-MEM culture medium with 1/100 volume of CMG 14–12 (equal to 10 ng/mL of M-CSF) and 100 ng/mL of recombinant RANKL for 4–5 days . 2.3. Tartrate-Resistant Acid Phosphatase (TRAP) Staining BMMs cultured in a 48-well plate were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 20 min at room temperature. TRAP was stained with NaK tartrate and Naphthol AS-BI phosphoric acid solution. The number of TRAP + multinucleated osteoclasts with more than 3 nuclei/well in four wells was counted and analyzed by one-way analysis of variance (ANOVA). 2.4. Real-Time Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) Total RNAs were purified using an RNeasy mini kit, and the first strand cDNAs were generated from 1 μg of total RNA using the High-Capacity cDNA Reverse Transcription Kit following the manufacturer’s instructions. Real-time qPCR was performed using the specific primers and TaqMan Gene Expression Master Mix in the QuantStudio3 real-time PCR system (Thermo-Fisher Scientific, Waltham, MA, USA) with an initial denaturation at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The relative cDNA amount was calculated by normalizing to that of the mitochondrial gene Mrps2 using the ΔCt method. 2.5. Lentiviral Transduction The recombinant lentiviruses expressing shRNAs targeting the murine Fam98b, Fam98c, and firefly luciferase (LUC) were generated. The 293-T cells were plated at a density of 0.75 × 10 6 cells/well in a 6-well tissue culture plate and cultured in DMEM medium containing 10% heat-inactivated FBS and 1 × PSG a day before transfection. The specific pLKO.1 gene transfer vectors (1.5 µg each) were co-transfected respectively with the 1.5 µg of lentivirus packing vectors, pCMV-delta-R8.2 and pMD2.G (at 8:1 ratio), using the TransIT-LT1 transfection reagent (9 µL in 100 µL of Opti-MEM/transfection). The medium was changed the next day. The virus supernatants were collected after 24 h and were filtrated through a 0.45 µm sterile nylon syringe filter (CAT# 76479-028, VWR, Radnor, PA, USA). For lentiviral transduction, 5 × 10 6 bone marrow cells were cultured in a 100 mm Petri dish for 3 days. The cells were then transduced with virus supernatant and 20 µg/mL of Protamine for 24 h. The BMMs were then selected in α-MEM culture medium containing 6 µg/mL puromycin for 3 days before use. 2.6. Immunoblotting Cells were lysed in RIPA buffer containing protease inhibitor cocktail. A total of 20 μg of proteins were separated by 8% SDS-polyacrylamide gel electrophoresis (PAGE). The proteins were detected with primary antibodies at 4 °C overnight, followed by incubation with HRP-conjugated anti-mouse secondary antibody. After rinsing three times with Tris-buffered saline containing 0.1% Tween 20, the membrane was subjected to enhanced chemiluminescent detection reagents used at 1:10 dilution. 2.7. Immunofluorescence The immunofluorescent staining was performed as described in our earlier work . The triple fluorescence-labeled osteoclasts were examined and analyzed using a Zeiss LSM 900 laser confocal scanning microscope run by Zeiss ZenBlue 3.1. 2.8. Resorption Pit Staining The resorption pit staining and quantification were conducted as described in our previous publications . 2.9. Generation of Fam98a-Flox and Fam98a-Flox;LysM-Cre Mice The Fam98a -flox mice on C57BL6/J background were commercially generated by Cyagen Biosciences. The Lyz2 -Cre (LysM-Cre) mice on C57BL6/J background (B6.129P2 tm1(cre)Ifo , CAT# 004781) were obtained from The Jackson Laboratory (Bar Harbor, ME, USA). The offspring of Fam98a -flox/+;LysM-Cre/Cre breeding pairs were genotyped for Fam98a -flox and Lyz2 -Cre using primers listed in the above table and from The Jackson Laboratory. The animal work follows the ARRIVE guidelines. All the in vivo and in vitro experiments were performed and analyzed in a double-blinded manner. 2.10. Micro-CT The femurs and L4 vertebrae isolated from 10-week-old male and female mice were cleaned of soft tissues and fixed in 4% paraformaldehyde in PBS for 3 days at 4 °C. The bones were imaged in a μCT (model vivaCT40; Scanco Medical AG, Wangen-Brüttisellen, Switzerland) with 55 to 70 kVp volts at a voxel size of 10.5 μm. A threshold of 200 was applied to all scans at medium resolution (E = 55 kVp, I = 145 μA, and integration time = 200 ms). 

The reagents used in this study were listed in . 

Whole bone marrow cells were extracted from the tibias and femurs of 10-week-old mice. Bone marrow monocytes (BMMs) and osteoclasts were cultured as described previously. In brief, red blood cells were lysed in 1× red blood cell lysis buffer for 5 min at room temperature. Bone marrow cells were plated onto a 100 mm Petri dish and cultured in α-MEM containing 10% heat-inactivated FBS, 1× Penicillin–Streptomycin-L-Glutamine (PSG), and 1/10 volume of CMG 14–12 (conditioned medium supernatant containing recombinant M-CSF at 1 μg/mL)  for 4 days. To generate osteoclasts from BMMs, 1.5 × 10 4 or 3 × 10 4 BMMs were cultured in α-MEM culture medium with 1/100 volume of CMG 14–12 (equal to 10 ng/mL of M-CSF) and 100 ng/mL of recombinant RANKL for 4–5 days . 

BMMs cultured in a 48-well plate were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 20 min at room temperature. TRAP was stained with NaK tartrate and Naphthol AS-BI phosphoric acid solution. The number of TRAP + multinucleated osteoclasts with more than 3 nuclei/well in four wells was counted and analyzed by one-way analysis of variance (ANOVA). 

Total RNAs were purified using an RNeasy mini kit, and the first strand cDNAs were generated from 1 μg of total RNA using the High-Capacity cDNA Reverse Transcription Kit following the manufacturer’s instructions. Real-time qPCR was performed using the specific primers and TaqMan Gene Expression Master Mix in the QuantStudio3 real-time PCR system (Thermo-Fisher Scientific, Waltham, MA, USA) with an initial denaturation at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The relative cDNA amount was calculated by normalizing to that of the mitochondrial gene Mrps2 using the ΔCt method. 

The recombinant lentiviruses expressing shRNAs targeting the murine Fam98b, Fam98c, and firefly luciferase (LUC) were generated. The 293-T cells were plated at a density of 0.75 × 10 6 cells/well in a 6-well tissue culture plate and cultured in DMEM medium containing 10% heat-inactivated FBS and 1 × PSG a day before transfection. The specific pLKO.1 gene transfer vectors (1.5 µg each) were co-transfected respectively with the 1.5 µg of lentivirus packing vectors, pCMV-delta-R8.2 and pMD2.G (at 8:1 ratio), using the TransIT-LT1 transfection reagent (9 µL in 100 µL of Opti-MEM/transfection). The medium was changed the next day. The virus supernatants were collected after 24 h and were filtrated through a 0.45 µm sterile nylon syringe filter (CAT# 76479-028, VWR, Radnor, PA, USA). For lentiviral transduction, 5 × 10 6 bone marrow cells were cultured in a 100 mm Petri dish for 3 days. The cells were then transduced with virus supernatant and 20 µg/mL of Protamine for 24 h. The BMMs were then selected in α-MEM culture medium containing 6 µg/mL puromycin for 3 days before use. 

Cells were lysed in RIPA buffer containing protease inhibitor cocktail. A total of 20 μg of proteins were separated by 8% SDS-polyacrylamide gel electrophoresis (PAGE). The proteins were detected with primary antibodies at 4 °C overnight, followed by incubation with HRP-conjugated anti-mouse secondary antibody. After rinsing three times with Tris-buffered saline containing 0.1% Tween 20, the membrane was subjected to enhanced chemiluminescent detection reagents used at 1:10 dilution. 

The immunofluorescent staining was performed as described in our earlier work . The triple fluorescence-labeled osteoclasts were examined and analyzed using a Zeiss LSM 900 laser confocal scanning microscope run by Zeiss ZenBlue 3.1. 

The resorption pit staining and quantification were conducted as described in our previous publications . 

The Fam98a -flox mice on C57BL6/J background were commercially generated by Cyagen Biosciences. The Lyz2 -Cre (LysM-Cre) mice on C57BL6/J background (B6.129P2 tm1(cre)Ifo , CAT# 004781) were obtained from The Jackson Laboratory (Bar Harbor, ME, USA). The offspring of Fam98a -flox/+;LysM-Cre/Cre breeding pairs were genotyped for Fam98a -flox and Lyz2 -Cre using primers listed in the above table and from The Jackson Laboratory. The animal work follows the ARRIVE guidelines. All the in vivo and in vitro experiments were performed and analyzed in a double-blinded manner. 

The femurs and L4 vertebrae isolated from 10-week-old male and female mice were cleaned of soft tissues and fixed in 4% paraformaldehyde in PBS for 3 days at 4 °C. The bones were imaged in a μCT (model vivaCT40; Scanco Medical AG, Wangen-Brüttisellen, Switzerland) with 55 to 70 kVp volts at a voxel size of 10.5 μm. A threshold of 200 was applied to all scans at medium resolution (E = 55 kVp, I = 145 μA, and integration time = 200 ms). 

3.1. All Three Murine Fam98 Family Genes Are Expressed in Precursor and Mature Osteoclasts In humans and mice, there are three FAM98 proteins (FAM98A, FAM98B, and FAM98C), which are encoded by distinct genes located on different chromosomes. FAM98A is the longest, and FAM98C is the shortest one among the three FAM98 proteins. The murine Fam98a has 19.6% identity and 22.7% similarity with both Fam98b and Fam98c ( ). Fam98b is closer to Fam98a than Fam98c, with 46.3% identity and 55.0% similarity, while Fam98c has only 25% identity and 33.4% similarity with Fam98a. To dissect the roles of FAM98s in osteoclasts, we first examined their mRNA expression during in vitro osteoclast differentiation from murine bone marrow monocytes (BMMs) by real-time quantitative RT-PCR. As shown in , the expression of Fam98c was found to be the highest among three genes in male and female osteoclast precursors and mature cells. The expression of Fam98b was slightly lower than that of Fam98a in osteoclast lineage cells. The mRNA expression of all three Fam98 genes decreased during the differentiation of both male and female osteoclasts. The protein level of Fam98s in murine osteoclast lineage cells was not assessed in this study because of a lack of reliable antibodies specifically recognizing each isoform of Fam98 family proteins. 3.2. Knockdown of Fam98c but Not Fam98b Expression Attenuates Osteoclastogenesis In Vitro We previously reported that Fam98a binds to Plekhm1, and the knockdown of Fam98a expression in vitro attenuates lysosome trafficking and secretion in murine osteoclasts . However, whether and how Fam98b and Fam98c regulate osteoclasts remain unknown. To unveil the role of Fam98b and Fam98c in osteoclast differentiation and/or function, we set out to knock down their expression in BMMs by lentiviral transduction of specific shRNA targeting Fam98b (Fam98b-sh) and Fam98c (Fam98c-sh), respectively. The expression of Fam98b-sh significantly reduced the mRNA level of Fam98b and led to a slight increase in Fam98a mRNA in BMMs relative to BMMs transduced with the control shRNA against firefly luciferase (LUC-sh) ( A), whereas Fam98c-sh expression in BMMs robustly decreased the amount of Fam98c mRNA but also resulted in a little decrease in Fam98a and Fam98b mRNAs compared to control BMMs ( A). We next cultured the different shRNA transduced BMMs with M-CSF and RANKL for 4 days to induce osteoclast differentiation. As shown by TRAP (tartrate-resistant acid phosphatase) staining and osteoclast number count ( B), the depletion of Fam98c greatly attenuated the formation of multinucleated osteoclasts (>3 nuclei/cell). Moreover, the mRNA and protein levels of Nfatc1, a master transcription factor of osteoclast differentiation, and the mRNA expression of osteoclast marker gene Acp5 (encodes TRAP), as well as the protein level of Cathepsin K, a crucial bone matrix-digesting enzyme in mature osteoclasts, decreased dramatically in Fam98c-suppressed osteoclast precursor and mature cells ( C,D). This result indicates that Fam98c is critical for osteoclastogenesis, probably because it regulates Nfatc1 expression in osteoclast lineage cells. By contrast, the repression of Fam98b expression in osteoclast precursors caused an increase in osteoclast number, as affirmed by higher levels of Nfatc1 and Acp5 in mononuclear and mature osteoclasts compared to control cells ( B–D). Of note, however, about 50% of Fam98b-knockdown osteoclasts (50.7 ± 8.4%, n = 4) displayed a similar morphological cell shape to Plekhm1 −/− osteoclasts with aggregated TRAP + lysosomes surrounding nuclei ( B, middle panel), suggesting that Fam98b, like Fam98a, may regulate osteoclast lysosome trafficking/secretion and bone resorption. 3.3. Decreased Expression of Fam98b in Osteoclasts Inhibits Lysosome Trafficking and Bone Resorption In Vitro To further define the function of Fam98b in osteoclasts, we cultured the control (LUC-sh) and Fam98b-knockdown (Fam98b-sh) BMMs with M-CSF and RANKL for 4 days on glass coverslips and cortical bovine bone slices, respectively. Lysosomes were labeled with a rat monoclonal antibody against murine Lamp-2, an integral membrane protein and marker of lysosomes in mammalian cells , co-stained with filament actin (F-actin) and nucleus. While a significant number of Lamp-2 + lysosomes were observed to be transported to and localized at the peripheral area inside the podosome ring in control osteoclasts cultured on glass coverslips, the lysosomes in Fam98b-deficient osteoclasts were aggregated around nuclei, void from the cell periphery ( A). In control osteoclasts cultured on bone slices, the Lamp-2 + lysosomes were targeted to the ruffled border membrane circumscribed by actin rings ( B, left panels). However, the Lamp-2 staining in Fam98b-deficient osteoclasts was mostly absent at the ruffled border ( B, right panels). Consistent with the important role of lysosome secretion in osteoclast bone resorption, the defective lysosome transportation in Fam98b-depressed osteoclasts led to a 50% reduction in bone resorption compared to control osteoclasts ( C). 3.4. Fam98a and Fam98b Compensate in Regulation of Lysosome Secretion and Bone Resorption in Osteoclasts To elucidate the role of FAM98A in the regulation of osteoclasts and bone homeostasis in vivo, we generated the Fam98a -flox mice in which the exons 4 and 5 of murine Fam98a gene were flanked by two loxP sites ( A). By crossing Fam98a -floxed mice with LysM-Cre mice, we created the Fam98a myeloid osteoclast precursor conditional knockout mice (cKO) ( A). The real-time quantitative PCR confirmed the deletion of exon 4 of the murine Fam98a gene in BMMs cultured from Fam98a cKO mice ( B). To maximize the deleting efficacy of Fam98a by LysM-Cre, we mated the Fam98a -flox/+; LysM-Cre/Cre breeding pairs to generate the control (Fam98a-+/+; LysM-Cre/Cre) and the Fam98a cKO (Fam98a-flox/flox;LysM-Cre/Cre) mice, similar to what we did in our recent published work . Unlike severe human osteopetrosis patients and mouse models , Fam98a cKO mice displayed no overt phenotypes in growth, body weight, craniofacial growth and development, and tooth eruption until 10 weeks of age. The femurs and lumber vertebrae were harvested from 10-week-old male and female mice for micro-CT analysis of bone mass and structures. As presented in C,D of micro-CT images and analyses, the trabecular bone volume (BV/TV) and cortical bone thickness (Cort.Th) in distal femurs of Fam98a cKO male and female mice were similar to those of control mice. The trabecular number (Tb.N), thickness (Tb.Th), and trabecular spacing (Tb.Sp) in Fam98a cKO male and female mice were indistinguishable from those of control mice. Additionally, there were no changes in the skeletal phenotypes of lumber vertebra in Fam98a cKO mice. These results indicate that loss of Fam98a in osteoclasts is dispensable for bone homeostasis in vivo. To unveil the impacts of loss of Fam98a in osteoclast precursors on osteoclastogenesis and bone resorption in vitro, we cultured the BMMs isolated from control and Fam98a cKO mice, respectively, with M-CSF and RANKL for 4 days on plastic culture dishes, glass coverslips, and cortical bovine bone slices. The number of TRAP + multinucleated osteoclasts and the mRNA expression of Nfatc1 and Acp5 in Fam98a-deficient BMMs, pre-osteoclasts, and mature osteoclasts were very close and indistinguishable from those in control cells ( A,B). In contrast to our previous findings in Fam98a shRNA knockdown osteoclasts , genetic ablation of Fam98a in osteoclasts had no effects on the peripheral distribution of Lamp-2 + lysosomes in osteoclasts cultured on glass coverslips and lysosome trafficking and targeting to the ruffled border in osteoclasts cultured on bone slices ( C,D). Thereafter, Fam98a −/− osteoclasts had a similar bone-resorbing capacity as compared to control osteoclasts ( E). Given that Fam98b is structurally and functionally related to Fam98a ( and ), it is likely that Fam98b may compensate for the loss of Fam98a in osteoclasts. To test if the lack of skeletal phenotype in Fam98a cKO mice can be explained by increased Fam98b expression, we first examined the mRNA expression of Fam98b and Fam98c in control and Fam98a −/− BMMs and pre-osteoclasts and mature osteoclasts. The expression of Fam98b but not Fam98c was upregulated in Fam98a −/− osteoclast precursor and mature cells compared to respective control osteoclasts ( A). Next, we transduced the Fam98a −/− BMMs with lentiviruses expressing the control (LUC-sh) and Fam98b-specific (Fam98b-sh) shRNAs, respectively. The mRNA level of Fam98b was dramatically and significantly reduced in Fam98b-sh transduced osteoclast precursor and mature cells ( B). The osteoclastogenesis from Fam98a −/− /Fam98b KD BMMs was comparable to the control BMMs with a slight increase in Nfatc1 but not Acp5 mRNA expression ( C,D) in Fam98a/b double-depleted osteoclast precursor and mature cells. However, the majority of Fam98a −/− /Fam98b KD osteoclasts (77 ± 12%, n = 4) exhibited irregular morphology with an accumulation of TRAP + lysosomes around nuclei ( C, lower panel) compared to Fam98b single knockdown osteoclasts (50.7 ± 8.4%, n = 4, in B). The severe defects in lysosome trafficking and bone resorption in Fam98a −/− /Fam98b KD osteoclasts were observed by Lamp-2 immunofluorescent and bone resorption pit staining ( E–G). These results postulate that Fam98a and Fam98b may compensate for each other in the regulation of lysosome secretion and bone resorption in osteoclasts in vitro and in vivo. This hypothesis warrants further investigation in the future. 

In humans and mice, there are three FAM98 proteins (FAM98A, FAM98B, and FAM98C), which are encoded by distinct genes located on different chromosomes. FAM98A is the longest, and FAM98C is the shortest one among the three FAM98 proteins. The murine Fam98a has 19.6% identity and 22.7% similarity with both Fam98b and Fam98c ( ). Fam98b is closer to Fam98a than Fam98c, with 46.3% identity and 55.0% similarity, while Fam98c has only 25% identity and 33.4% similarity with Fam98a. To dissect the roles of FAM98s in osteoclasts, we first examined their mRNA expression during in vitro osteoclast differentiation from murine bone marrow monocytes (BMMs) by real-time quantitative RT-PCR. As shown in , the expression of Fam98c was found to be the highest among three genes in male and female osteoclast precursors and mature cells. The expression of Fam98b was slightly lower than that of Fam98a in osteoclast lineage cells. The mRNA expression of all three Fam98 genes decreased during the differentiation of both male and female osteoclasts. The protein level of Fam98s in murine osteoclast lineage cells was not assessed in this study because of a lack of reliable antibodies specifically recognizing each isoform of Fam98 family proteins. 

We previously reported that Fam98a binds to Plekhm1, and the knockdown of Fam98a expression in vitro attenuates lysosome trafficking and secretion in murine osteoclasts . However, whether and how Fam98b and Fam98c regulate osteoclasts remain unknown. To unveil the role of Fam98b and Fam98c in osteoclast differentiation and/or function, we set out to knock down their expression in BMMs by lentiviral transduction of specific shRNA targeting Fam98b (Fam98b-sh) and Fam98c (Fam98c-sh), respectively. The expression of Fam98b-sh significantly reduced the mRNA level of Fam98b and led to a slight increase in Fam98a mRNA in BMMs relative to BMMs transduced with the control shRNA against firefly luciferase (LUC-sh) ( A), whereas Fam98c-sh expression in BMMs robustly decreased the amount of Fam98c mRNA but also resulted in a little decrease in Fam98a and Fam98b mRNAs compared to control BMMs ( A). We next cultured the different shRNA transduced BMMs with M-CSF and RANKL for 4 days to induce osteoclast differentiation. As shown by TRAP (tartrate-resistant acid phosphatase) staining and osteoclast number count ( B), the depletion of Fam98c greatly attenuated the formation of multinucleated osteoclasts (>3 nuclei/cell). Moreover, the mRNA and protein levels of Nfatc1, a master transcription factor of osteoclast differentiation, and the mRNA expression of osteoclast marker gene Acp5 (encodes TRAP), as well as the protein level of Cathepsin K, a crucial bone matrix-digesting enzyme in mature osteoclasts, decreased dramatically in Fam98c-suppressed osteoclast precursor and mature cells ( C,D). This result indicates that Fam98c is critical for osteoclastogenesis, probably because it regulates Nfatc1 expression in osteoclast lineage cells. By contrast, the repression of Fam98b expression in osteoclast precursors caused an increase in osteoclast number, as affirmed by higher levels of Nfatc1 and Acp5 in mononuclear and mature osteoclasts compared to control cells ( B–D). Of note, however, about 50% of Fam98b-knockdown osteoclasts (50.7 ± 8.4%, n = 4) displayed a similar morphological cell shape to Plekhm1 −/− osteoclasts with aggregated TRAP + lysosomes surrounding nuclei ( B, middle panel), suggesting that Fam98b, like Fam98a, may regulate osteoclast lysosome trafficking/secretion and bone resorption. 

To further define the function of Fam98b in osteoclasts, we cultured the control (LUC-sh) and Fam98b-knockdown (Fam98b-sh) BMMs with M-CSF and RANKL for 4 days on glass coverslips and cortical bovine bone slices, respectively. Lysosomes were labeled with a rat monoclonal antibody against murine Lamp-2, an integral membrane protein and marker of lysosomes in mammalian cells , co-stained with filament actin (F-actin) and nucleus. While a significant number of Lamp-2 + lysosomes were observed to be transported to and localized at the peripheral area inside the podosome ring in control osteoclasts cultured on glass coverslips, the lysosomes in Fam98b-deficient osteoclasts were aggregated around nuclei, void from the cell periphery ( A). In control osteoclasts cultured on bone slices, the Lamp-2 + lysosomes were targeted to the ruffled border membrane circumscribed by actin rings ( B, left panels). However, the Lamp-2 staining in Fam98b-deficient osteoclasts was mostly absent at the ruffled border ( B, right panels). Consistent with the important role of lysosome secretion in osteoclast bone resorption, the defective lysosome transportation in Fam98b-depressed osteoclasts led to a 50% reduction in bone resorption compared to control osteoclasts ( C). 

To elucidate the role of FAM98A in the regulation of osteoclasts and bone homeostasis in vivo, we generated the Fam98a -flox mice in which the exons 4 and 5 of murine Fam98a gene were flanked by two loxP sites ( A). By crossing Fam98a -floxed mice with LysM-Cre mice, we created the Fam98a myeloid osteoclast precursor conditional knockout mice (cKO) ( A). The real-time quantitative PCR confirmed the deletion of exon 4 of the murine Fam98a gene in BMMs cultured from Fam98a cKO mice ( B). To maximize the deleting efficacy of Fam98a by LysM-Cre, we mated the Fam98a -flox/+; LysM-Cre/Cre breeding pairs to generate the control (Fam98a-+/+; LysM-Cre/Cre) and the Fam98a cKO (Fam98a-flox/flox;LysM-Cre/Cre) mice, similar to what we did in our recent published work . Unlike severe human osteopetrosis patients and mouse models , Fam98a cKO mice displayed no overt phenotypes in growth, body weight, craniofacial growth and development, and tooth eruption until 10 weeks of age. The femurs and lumber vertebrae were harvested from 10-week-old male and female mice for micro-CT analysis of bone mass and structures. As presented in C,D of micro-CT images and analyses, the trabecular bone volume (BV/TV) and cortical bone thickness (Cort.Th) in distal femurs of Fam98a cKO male and female mice were similar to those of control mice. The trabecular number (Tb.N), thickness (Tb.Th), and trabecular spacing (Tb.Sp) in Fam98a cKO male and female mice were indistinguishable from those of control mice. Additionally, there were no changes in the skeletal phenotypes of lumber vertebra in Fam98a cKO mice. These results indicate that loss of Fam98a in osteoclasts is dispensable for bone homeostasis in vivo. To unveil the impacts of loss of Fam98a in osteoclast precursors on osteoclastogenesis and bone resorption in vitro, we cultured the BMMs isolated from control and Fam98a cKO mice, respectively, with M-CSF and RANKL for 4 days on plastic culture dishes, glass coverslips, and cortical bovine bone slices. The number of TRAP + multinucleated osteoclasts and the mRNA expression of Nfatc1 and Acp5 in Fam98a-deficient BMMs, pre-osteoclasts, and mature osteoclasts were very close and indistinguishable from those in control cells ( A,B). In contrast to our previous findings in Fam98a shRNA knockdown osteoclasts , genetic ablation of Fam98a in osteoclasts had no effects on the peripheral distribution of Lamp-2 + lysosomes in osteoclasts cultured on glass coverslips and lysosome trafficking and targeting to the ruffled border in osteoclasts cultured on bone slices ( C,D). Thereafter, Fam98a −/− osteoclasts had a similar bone-resorbing capacity as compared to control osteoclasts ( E). Given that Fam98b is structurally and functionally related to Fam98a ( and ), it is likely that Fam98b may compensate for the loss of Fam98a in osteoclasts. To test if the lack of skeletal phenotype in Fam98a cKO mice can be explained by increased Fam98b expression, we first examined the mRNA expression of Fam98b and Fam98c in control and Fam98a −/− BMMs and pre-osteoclasts and mature osteoclasts. The expression of Fam98b but not Fam98c was upregulated in Fam98a −/− osteoclast precursor and mature cells compared to respective control osteoclasts ( A). Next, we transduced the Fam98a −/− BMMs with lentiviruses expressing the control (LUC-sh) and Fam98b-specific (Fam98b-sh) shRNAs, respectively. The mRNA level of Fam98b was dramatically and significantly reduced in Fam98b-sh transduced osteoclast precursor and mature cells ( B). The osteoclastogenesis from Fam98a −/− /Fam98b KD BMMs was comparable to the control BMMs with a slight increase in Nfatc1 but not Acp5 mRNA expression ( C,D) in Fam98a/b double-depleted osteoclast precursor and mature cells. However, the majority of Fam98a −/− /Fam98b KD osteoclasts (77 ± 12%, n = 4) exhibited irregular morphology with an accumulation of TRAP + lysosomes around nuclei ( C, lower panel) compared to Fam98b single knockdown osteoclasts (50.7 ± 8.4%, n = 4, in B). The severe defects in lysosome trafficking and bone resorption in Fam98a −/− /Fam98b KD osteoclasts were observed by Lamp-2 immunofluorescent and bone resorption pit staining ( E–G). These results postulate that Fam98a and Fam98b may compensate for each other in the regulation of lysosome secretion and bone resorption in osteoclasts in vitro and in vivo. This hypothesis warrants further investigation in the future. 

Three FAM98 family proteins (FAM98A/B/C) have been identified in humans and mice based on sharing a unique structure domain with unknown function (DUF2465). By bioinformatic profiling and structural modeling, this domain is also found in several microtubule-associated proteins, including the intraflagellar transport (IFT) complex B subunits (IFT81, IFT57, and CLUAP1), suggesting a hypothetic role of FAM98 family proteins in microtubule dynamics and/or microtubule-based transportations . FAM98A and FAM98B have been shown to work in an RNA-binding complex, positively regulating RNA transcription, tRNA splicing, and RNA translation [ , , , , ]. FAM98C has been identified as a candidate gene linked to ciliogenesis . Nonetheless, the expression and physiological functions of FAM98A/B/C in bone cells and skeleton modeling/remodeling have not been fully elucidated and remain largely unknown. We have previously reported that Fam98a interacts with Plekhm1 in murine osteoclasts and suppression of Fam98a expression by shRNA-mediated specific knockdown of Fam98a mRNA in osteoclasts attenuates osteoclast lysosome trafficking and bone resorption, indicating that FAM98A might couple lysosomes to microtubules via its binding to lysosomal adaptor protein PLEKHM1 . Unexpectedly, however, we found in this study that loss of Fam98a by its genetic deletion in osteoclast myeloid precursor cells was dispensable for osteoclastogenesis and osteoclast function in vitro as well as bone modeling/remodeling in vivo. Since the knockdown of Fam98b expression by shRNA in control and Fam98a −/− osteoclasts exhibited defects in lysosome trafficking and bone resorption and since Fam98b expression was higher in Fam98a −/− osteoclasts than in control cells, it is likely that Fam98b compensates for the loss of function of Fam98a in osteoclasts. Fam98b retains more structural similarity with Fam98a than Fam98c, and FAM98A/B has been shown to function redundantly in colorectal cancer cells . As constituted in a protein complex regulating RNA metabolism and translation, FAM98A/B may activate PLEKHM1 mRNA maturation and/or translation. Thereby, suppression of FAM98A/B may cause a decreased level of PLEKHM1, which is indispensable for lysosome trafficking and bone resorption in osteoclasts . Unfortunately, there is no reliable and high-quality antibody available to detect endogenous PLEKHM1 to test this hypothesis. Alternatively, FAM98A/B may anchor lysosomes onto microtubules via its interaction with PLEKHM1 and the N-terminal conserved DUF2465 domain. Nevertheless, the underlying mechanisms by which FAM98A/B regulates osteoclast lysosome trafficking and bone resorption warrant further investigation in the future. The mRNA level of Fam98c was the highest among the three Fam98 genes in cultured murine osteoclast precursor and mature cells, as detected by RT-qPCR in the present study. Moreover, repression of Fam98c but not loss of Fam98a/b greatly reduced the mRNA and protein levels of the master osteoclast differentiation transcription factor Nfatc1 and dramatically inhibited osteoclastogenesis in vitro. These results indicate that FAM98C plays a critical role in osteoclast formation, at least in vitro. The exact mechanisms by which FAM98C specifically regulates osteoclast differentiation and/or survival are unknown. One possibility is that FAM98C may be involved in NFATC1 mRNA processing and translation since FAM98C has been linked to ciliogenesis  and the primary cilium has been recently reported to regulate osteoclastogenesis . It is also likely that FAM98C regulates osteoclast differentiation through its action on the primary cilia dynamics. However, it should be noted that the important role of primary cilium in the regulation of osteoclasts has not yet been well established compared to osteoblasts and osteocytes  and requires more future research. Although our work so far has uncovered unique roles of FAM98 family proteins in osteoclastogenesis and osteoclast bone resorption, there are several limitations of the current study. First, the detailed and comprehensive mechanisms by which FAM98A/B and FAM98C regulate lysosome secretion/bone resorption and osteoclastogenesis, respectively, have not been elucidated and need further investigations in the future. Moreover, the compensatory changes of other components of lysosome-trafficking machinery in FAM98A/B-deficient osteoclasts have not been executed and require further studies. Second, FAM98A and FAM98B have been implicated in tumorigenesis and progression. Whether and how FAM98A/B regulates lysosome secretion and degradation of extracellular matrix in cancer cells have not been conducted in this study. In addition, whether and how the downregulation of FAM98A and FAM98B plays a role in osteoclast-mediated cancer bone metastasis need studies using relevant animal models. Third, the functions of FAM98 family proteins under pathological conditions and in human disease-relevant animal models have not been carried out in this study. Such investigations will significantly improve the clinical and translational implications of targeting FAM98 family proteins as novel therapeutic strategies for the treatment of metabolic bone diseases and cancers. 

FAM98 family proteins play distinct roles in osteoclastogenesis and osteoclast bone resorption, respectively. FAM98C plays an important role in osteoclastogenesis, whereas FAM98A and FAM98B specifically regulate osteoclast lysosome trafficking and bone resorption. 

