CTRP4: a new member of the adipocytokine family

Human CTRP4 is a member of the C1q/TNF-related protein (CTRP) superfamily. This family, originally identified by Lodish and coworkers, is a highly conserved family consisting of secreted proteins that were cloned on the basis of an adiponectin homology sequence.¹ Up to now, 15 members have been identified in CTRP superfamily (CTRP1–CTRP15) (Figure 1). Those, together with adiponectin, leptin and resistin, are collectively known as the adipocytokine superfamily.² CTRPs usually assume a homotrimeric structure and each subunit is composed of an N-terminal signal peptide, a short variable region, a collagen domain and a C-terminal C1q globular domain.³ Accumulating evidence has demonstrated that CTRPs play important regulatory roles in inflammation, metabolism and tumor angiogensis.

Figure 1

Structure of CTRP4 and its effects on metabolism, inflammation and cancer.

We first reported the cloning of CTRP4 through bioinformatic analysis and high-throughput screening in 2011.⁴ Using a NF-κB luciferase reporter gene screening, we found that CTRP4 was a potent activator of NF-κB in 293T, HeLa and HepG-2 cells. CTRP4 contains a classical N-terminal signal peptide, suggesting that it may be secreted as a cytokine. In support, recombinant CTRP4 can effectively activate both NF-κB and IL-6/STAT3 signaling pathways and promote the secretion of IL-6 and TNF.

In contrast to a single C1q globular domain in other members of the CTRP4 superfamily, two such domians are found in CTRP4. They share 51% homology to each other, while the similarity is only 27–30% between them and the complement component C1q.⁵ CTRP4 appears to be well conserved in evolution since human and mouse CTRP4 share over 90% homology in amino-acid sequence. As much as its expression pattern is concerned, CTRP4 is mainly detected in brain, fat and bone marrow stem cells. In addition, it is present in the blood as a circulating protein.

In order to explore its physiological functions, we have prepared both polyclonal and monoclonal antibodies against CTRP4. Moreover, a mouse strain was also generated for transgenic expression of CTRP4.^{6, 7, 8} Contradictory to the findings from in vitro studies, in vivo experiments with various inflammatory models have demonstrated that CTRP4 primarily exerted an anti-inflammatory activity. In the DSS-induced mouse colitis model,⁹ transgenic expression of CTRP4 significantly relieved the symptoms and prolonged the survival of mice. Similar attenuating effects were also observed after administration of recombinant CTRP4. In an azoxymethane (AOM)/repeated administration of dextran sodium sulfate (DSS)-induced inflammatory cancer model, CTRP4 transgenic mice displayed resistance to tumorigenesis.¹⁰ Fewer transgenic mice developed colonic tumors and the tumors arising in the transgenic mice had smaller volume and were mainly adenoma instead of adenocarcinoma. Presumably, the reduced occurrence of tumor might be related to suppressed inflammation. Of note, NF-κB and STAT3 activation was significantly reduced in colitis specimens from the CTRP4 transgenic mice.¹¹

The CTRP4’s roles in promoting tumor cell proliferation in vitro and suppressing inflammation and tumorigenesis in vivo indicate that CTRP4 may have different functions depending on cell types, receptors and/or cell context. In the effort of searching for the cell types that mediate CTRP4’s anti-inflammatory role, we found that rhCTRP4 treatment suppressed Lipopolysaccharide-induced STAT3 and NF-kB activation and pro-inflammatory cytokine production in bone marrow-derived macrophages. Thus, the anti-inflammatory role of CTRP4 can be at least realized by directly or indirectly suppressing macrophage activation in response to pathogen-associated molecular patterns.

As a member of adipocytokine superfamily, our results further indicate that CTRP4 is an important regulator in glucose and lipid metabolism. CTRP4 transgenic mice showed higher level of food intake but were highly resistant to obesity and hyperglycemia. Compared to wild-type mice, CTRP4 transgenic mice had lower insulin resistance and liver fat, and reduced infiltration of inflammatory cells in adipose tissue (unpublished results). The study by Byerly et al. also found that central administration of recombinant CTRP4 protein altered the whole-body energy balance in both chow-fed and high-fat diet-fed mice. Different from our results, Byerly found reduced food intake in the group receiving CTRP4 protein in the brain, and decreased expression of orexigenic neuropeptide (Npy and Agrp) genes in the hypothalamus was associated with CTRP4-mediated food intake reduction.¹² The disparity between Byerly’s and our results suggest that CTRP4 produced by tissues other than brain may also affect food intake. Taken together, the results from Byerly and our groups demonstrate that CTRP4 is an important regulator of metabolism and energy balance.

In summary, the results from transgenic mice and recombinant protein administration in vivo have demonstrated that CTRP4 is an adipocytokine with important functions in negative regulation of inflammation and metabolism. Future studies with CTRP4 loss-of-function mice will be important to investigate the underlying mechanisms of CTRP4 and to uncover other functions of this evolutionarily conserved protein. Another key question to date, it remains elusive of the receptor that binds to and mediates the activities of CTRP4. Identification of the receptor and dissection of the downstream signaling will be critical to fully appreciate its roles under physiological and pathological conditions. There may be more than one receptor that can bind to CTRP4 as it could activate NF-B and STAT3 in cancer cell line but suppress these signaling pathways in macrophages. The identification of the specific receptors will facilitate the functional studies of CTRP4 and even the entire CTRP superfamily.

Accumulating evidence indicates that CTRP4 possesses multiple important functions. Studies by ours and others have demonstrated that the CTRP4 recombinant protein presents good biological activity both in vitro and in vivo, highlighting its potential as a prospective therapeutic agent.¹³