Members of the SCUBE family, such as SCUBE3, are secreted, cell surface glycoproteins with a characteristic structure that includes an N-terminal signal peptide followed by multiple EGF (131530)-like repeats and a C-terminal CUB domain. SCUBE3 is highly expressed in primary osteoblasts and bone (Wu et al., 2004).

By searching databases for sequences similar to SCUBE1 (611746) and SCUBE2 (611747), followed by PCR of primary human osteoblasts, Wu et al. (2004) cloned SCUBE3. The deduced 993-amino acid protein has an N-terminal signal sequence, followed by 9 putative calcium-binding EGF-like repeats, a spacer region, and a C-terminal CUB domain. The spacer region contains 5 potential N-glycosylation sites. SCUBE3 was predicted to be secreted as a mature 971-amino acid protein with a calculated molecular mass of 107 kD. Wu et al. (2004) also identified mouse Scube3, which shares over 96% amino acid identity with human SCUBE3. Northern blot analysis detected an 8-kb SCUBE3 transcript that was highly expressed in primary human osteoblasts. Lower expression of a 4-kb transcript was detected in umbilical vein endothelial cells, and the 4-kb transcript was also weakly expressed in adult heart. SCUBE3 was not detected in other adult human tissues. SCUBE3 was secreted from transfected HEK293T cells. The secreted protein had an apparent molecular mass of 130 kD, which was reduced to about 110 kD following inhibition of N-glycosylation.

Wu et al. (2011) noted the presence of several cysteine-rich motifs within the spacer region of SCUBE3 just prior to the CUB domain.

By quantitative RT-PCR analyses in newborn mice, Lin et al. (2021) demonstrated that Scube3 is highly expressed in long bone tissues. Immunohistochemical staining revealed that Scube3 is specifically expressed in the periosteum and trabecular endosteum, where osteoprogenitor cells and osteoblasts reside. High expression levels were also observed in proliferative/prehypertrophic chondrocytes of the growth plate; the authors noted that BMP2 (112261) and BMP4 (112262) are also highly expressed in the growth plate, suggesting a role for SCUBE3 in BMP-mediated chondrogenesis and/or osteoblastogenesis.

By genomic sequence analysis, Wu et al. (2004) mapped the SCUBE3 gene to chromosome 6p21.3. They mapped the mouse Scube3 gene to a region of chromosome 17B that shares homology of synteny with human chromosome 6p21.3.

Using immunoprecipitation and Western blot analysis, Wu et al. (2004) found that epitope-tagged SCUBE3 could form homooligomers and heterooligomers with SCUBE1 following overexpression in HEK293T cells. SCUBE3 also appeared to undergo limited proteolysis following secretion. The processed protein had an apparent molecular mass of about 65 kD. This form appeared to result from cleavage at a furin (FUR; 136950)-like cleavage site in the spacer region of SCUBE3.

By EST database analysis, Wu et al. (2011) found that SCUBE3 was overexpressed in lung tumors compared with normal lung. RT-PCR analysis and histologic examination of lung tumors and adjacent normal tissues confirmed the observation. In lung cancer cell lines, the degree of SCUBE3 expression correlated positively with cell line invasiveness. The C-terminal CUB domain of SCUBE3, which could be released from full-length SCUBE3 by MMP2 (120360) and MMP9 (120361) in vitro, bound to TGF type II receptor (TGFBR2; 190182), activated TGF-beta-1 (TGFB1; 190180)-like signaling, induced SMAD2 (601366)/SMAD3 (603109) phosphorylation and transcriptional activity, and caused epithelial-mesenchymal transition in transfected cells. Cotreatment with TGF-beta-1 and the SCUBE3 CUB domain enhanced expression of a SMAD2/SMAD3 reporter, suggesting that SCUBE3 and TGF-beta-1 do not compete for receptor binding. Knockdown of SCUBE3 expression suppressed cancer cell migration and invasiveness in lung tumor cell lines in culture and suppressed tumorigenesis and cancer metastasis following injection into nude mice.

To assess whether SCUBE3 interacts with ligands and receptors of the BMP signaling pathway, Lin et al. (2021) performed coimmunoprecipitation assays using lysates of HEK293T cells transfected to overexpress SCUBE3 with BMP2, BMP4, and BMP7 (112267), the 3 major BMP ligands implicated in early limb patterning and skeletogenesis. Immunoprecipitation followed by immunoblotting documented coprecipitation of SCUBE3 with BMP2 and BMP4, and slightly reduced coimmunoprecipitation with BMP7. Further experiments demonstrated that SCUBE3 coimmunoprecipitated with BMPR1A (601299) and BMPR1B (603248) in a ligand-dependent manner, suggesting a potential BMP ligand-inducible association between SCUBE3 and BMPR1A or 1B. To investigate the effect of SCUBE3 on BMP2/4-stimulated osteoblast differentiation, the authors measured induced alkaline phosphatase (ALP) activity and phosphorylation status of Smad1 (601595)/5 (603110)/8 (603295) in the mouse mesenchymal progenitor cell line C3H10T1/2. BMP2-, BMP4-, and BMP7-induced ALP activity and downstream activation of Smad1/5/8 were significantly and concomitantly suppressed by Scube3 knockdown. In addition, ectopic SCUBE3 overexpression in C3H10T1/2 cells resulted in a marked induction of ALP activity as well as enhanced BMP2/4-induced phosphorylation of Smad1/5/8. The authors concluded that SCUBE3 may play a positive modulatory role on BMP2/4 signaling, exerting osteogenic function by modulating BMP-stimulated osteoblast differentiation. Noting that BMP receptor association with membrane microdomains (lipid rafts) is a critical event for BMP signaling and that overexpressed SCUBE3 is a raft-associated protein, Lin et al. (2021) also examined raft recruitment of BMP receptors in mouse chondrocytes. BMP-induced raft association of BMP receptors was completely abolished in Scube3 -/- chondrocytes, indicating a requirement of SCUBE3 for the stimulus-dependent redistribution of receptors. The authors suggested that SCUBE3 facilitates BMP2-induced BMP receptor association with membrane microdomains.

In affected individuals from 9 unrelated consanguineous families with short stature, facial dysmorphism, and skeletal anomalies with or without cardiac anomalies (SSFSC2; 619184), Lin et al. (2021) identified homozygosity for 8 different mutations in the SCUBE3 gene, including missense, nonsense, frameshift, and splicing changes, as well as a complex intragenic arrangement (see, e.g., 614708.0001-614708.0005).

Fuchs et al. (2016) generated a mutant mouse line with an N294K missense mutation in exon 8 of the Scube3 gene and observed reduced body size, a shorter and kinky tail, abnormal digit positioning, and an abnormal posture when hung by the tail. X-ray analysis of the skeleton revealed malformations of the thoracic and lumbar vertebrae and shorter femora compared to wildtype mice. Skeletal staining at various embryonic and postnatal stages showed multiple hyperossification centers, with ossification centers and pedicles that were more closed and fused compared to wildtype mice. Hyperossification was detected at embryonic day 17.5, after the start of endochondral ossification, suggesting a defect in bone growth rather than disturbed embryonic patterning. The mutant mice also had disturbances in renal function and hearing deficits, and showed altered energy metabolism, with significantly reduced body mass and lower absolute fat and lean mass compared to wildtype mice.

Lin et al. (2021) generated Scube3 -/- mice and observed reduced growth, associated with smaller forelimbs and hindlimbs. In addition, the Scube3-null mice frequently showed misaligned upper and lower incisors as well as craniofacial defects, including a shorter and narrower face, smaller forehead, and reduced frontonasal and mandibular regions. Some mice also showed a protruding rib cage or hunchback spine. Histologic and immunohistochemical analysis of embryonic and postnatal long bones from Scube3 -/- mice indicated that Scube3 contributes to fine-tuning BMP-dependent endochondral bone formation by regulating both chondrogenic differentiation and chondrocyte proliferation as well as osteogenic differentiation.