Potential conflict of interest: Nothing to report.
The matrix metalloproteinase (MMP) family of proteins degrades extracellular matrix (ECM) components as well as processes cytokines and growth factors. MMPs are involved in regulating ECM homeostasis in both normal physiology and disease pathophysiology. Here we report the critical roles of mmp23b in normal zebrafish liver development. Mmp23b was initially identified as a gene linked to the genomic locus of an enhancer trap transgenic zebrafish line in which green fluorescent protein (GFP) expression was restricted to the developing liver. Follow-up analysis of mmp23b messenger RNA (mRNA) expression confirmed its liver-specific expression pattern. Morpholino knockdown of mmp23b resulted in defective hepatocyte proliferation, causing a reduction in liver size while maintaining relatively normal pancreas and gut development. Genetically, we showed that mmp23b functions through the tumor necrosis factor (TNF) signaling pathway. Antisense knockdown of tnfa or tnfb in zebrafish caused similar reductions of liver size, whereas overexpression of tnfa or tnfb rescued liver defects in mmp23b morphants but not vice versa. Biochemically, MMP23B, the human ortholog of Mmp23b, directly interacts with TNF and mediates its release from the cell membrane in a cell culture system. Because mmp23b/MMP23B is highly conserved, our findings in zebrafish warrant further investigation of its role in regulating liver development in mammals. (HEPATOLOGY 2010)
Matrix metalloproteinases (MMPs) are a family of proteinase that degrade the extracellular matrix (ECM) by substrate cleavage,1 regulating physiological processes during embryonic development, tissue remodeling, and disease processes such as metastasis, arthritis, and liver fibrosis.2, 3 Hepatic fibrosis is characterized by increased deposition, altered composition of the ECM, and regenerative nodule formation.4 In response to injury, increased proinflammatory cytokines induce MMP expression in hepatocytes including hepatic stellate cells (HSC).4 For example, levels of MMP-13, MMP-2, MMP-9, MT1-MMP, MMP-3, and MMP-10 are all increased during liver injury and fibrosis.5 MMPs degrade normal ECM in the space of Disse, which in turn activates HSC transdifferentiation into myofibroblasts producing fibrillar, contractile ECM, whereas the remaining HSC population undergoes apoptosis.4
In addition to being responsible for the turnover and degradation of connective-tissue proteins, MMPs regulate the activity of cytokines and chemokines by substrate cleavage.6, 7 One MMP-regulated proinflammatory cytokine is tumor necrosis factor (TNF).8 Hepatic TNF is mainly derived from Kupffer cells and regulates liver homeostasis by modulating hepatocyte proliferation as well as cell death.9, 10 TNF has also been implicated in the pathogenesis of alcoholic and nonalcoholic fatty liver injury as well as inflammation response during liver fibrosis in human and lower vertebrates.11, 12 Systemic administration of TNF induces hepatocyte proliferation.13 In TNF-R1-deficient animal models, hepatocyte DNA synthesis is severely impaired, establishing that TNF can initiate hepatocyte proliferation in response to tissue loss.14 TNF is produced as a 26-kDa membrane-bound protein (proTNF) that is cleaved into a soluble 17-kDa cytokine by TNF-converting enzyme transarterial chemoembolization (TACE/ADAM17),15, 16 a member of the disintegrin family of metalloproteinases (ADAMs). MMPs have also been shown to be capable of processing TNF to its soluble form,17–19 but no known MMPs can cleave proTNF as effectively as TACE.20
Compared to their role in liver fibrosis, MMP functions during normal liver development are not well known. The regulation of ECM remodeling during morphogenesis is poorly understood because of the lack of good in vivo models for the study of MMP regulation during embryonic development. Improved understanding of ECM remodeling may also improve the understanding of the pathological mechanisms underlying the misregulation of ECM activity in tumors.21 A recent study by Wyatt et al.21 developed several elaborate techniques for studying MMPs in zebrafish (Daniorerio), demonstrating the zebrafish embryo as a more tractable experimental organism to study the regulation of MMP activity during development.
Zebrafish have a conserved liver developmental process that shares striking similarity to mammals.22 The optical clarity during embryogenesis allows visualization of the entire liver developmental process and morphology if a transgenic green or red fluorescent protein (GFP or RFP) reporter is expressed in the liver. We performed a large-scale Tol2-based enhancer trap screen for transgenic zebrafish exhibiting tissue-specific GFP expression and mapped 15 lines with liver expression patterns to specific trapped genes. One such line, mp255c, trapped a gene designated matrix metalloproteinase 23b (mmp23b); mmp23b is orthologous to mammalian MMP23B, which has no described function. A recent microarray study using a zebrafish liver cancer model showed that mmp23b is significantly down-regulated in liver cancers, whereas other MMPs are increased (23), implying the uniqueness of this MMP member in liver biology. We show that endogenous mmp23b is expressed in hepatocytes during development and its proper function is required for hepatocyte proliferation. Genetically, mmp23b functions through TNFs. Evidence in vitro suggests an MMP23B-mediated protein shedding mechanism might be involved in regulating TNF activity. Because mmp23b/MMP23B is highly conserved, its role in liver development in mammals should be further studied.
More reagents and procedures used in this report are described in detail in the Supporting Materials and Methods.
Zebrafish were raised and kept under standard laboratory conditions at ≈28°C. Embryos were staged according to Kimmel et al.24Mmp23b:GFP fish were generated from a previous Tol2-based enhancer trap screen. The wildtype line used was AB.
RNA Whole-Mount In Situ Hybridization (WISH).
WISH was performed essentially as described.25 NBT/BCIP (50 mg/mL; Promega) were used as alkaline phosphatase substrates. The following probes were used: mmp23b,ceruloplasmin,hhex, gata6, insulin, and carboxypeptidaseA.
RNA Synthesis and Injection.
Mmp23b,tnfa, and tnfb full-length coding sequences were obtained from Open Biosystems and subcloned into a pCS2+ vector. Messenger RNAs (mRNAs) were synthesized using T7 or SP6 mMessage mMachine kit (Ambion). mRNA injection was performed as described at the one-cell stage.26 Sterile water was used for the control experiments. One hundred picograms of mmp23b,tnfa and tnfb mRNA was used for all experiments.
Antisense MO Oligonucleotides.
MOs designed against mmp23b targeting either the ATG (5′-AGCA CACGAACAAACCCAGAACATC-3′) or 5′-UTR (5′-TGAAATCACAACTTTCCTCACGGAT-3′) was obtained from Gene Tools. Tnfa-MO1 (5′-GGCAGG ATTTTCACCTTATGGAGCG-3′) and tnfb-MO1 (5′-AATTTCAGTCTTACCATCACATGCC-3′) were obtained from Open Biosystems. A standard MO control oligo was obtained from Gene Tools. All MOs were resuspended in sterile water at 2 ng/nl and injected into one- to two-cell stage embryos. More sequence information is provided in the Supporting Materials.
Whole-Mount Immunostaining and Immunohistochemistry.
Whole-mount immunostaining with phospho-histone H3 (pH3) antibody was performed as described.27Mmp23b:GFP transgenic fish were used for double staining. Primary antibodies were used as follows: rabbit anti-pH3 Ser 10 (Santa Cruz) at 1:400, mouse anti-GFP (Invitrogen) at 1:200. Alexa Fluor 488-conjugated antimouse IgG and Alexa Fluor 568-conjugated goat antirabbit IgG were used as secondary antibodies (Invitrogen). The whole embryos were then observed with confocal microscopy. Preparation of paraffin sections and cryosections for immunohistochemistry is described in the Supporting Materials.
Cell Culture and Transfection.
Mtace (gift from Dr. R. Black) and HMMP23B (Open Biosystems) full-length coding sequences were subcloned into pcDNA3.1+ (Invitrogen) vector. Murine TACE−/− fibroblasts were gifts from Dr. R. Black and were cultured as described.28 Transfection and samples collection are described in the Supporting Materials.
Western Blots and Enzyme-Linked Immunosorbent Assay (ELISA) Detection.
Western blotting was performed using a standard protocol; anti-FLAG was obtained from Sigma; anti-tubulin (Invitrogen) was used as a loading control. Cell lysates were measured for TNF content by ELISA as described29 and normalized to total protein using a protein assay (Pierce).
Tol2 Transposon-Based Enhancer Trap Screen.
We performed a large-scale enhancer trap screen in zebrafish using Tol2 transposon-mediated transgenic approaches.30 The Tol2GT2MP constructs are modified Tol2 mobile elements created for the purpose of enhancer trapping in zebrafish. They carry a minimal promoter fragment from the zebrafish gata2 gene31 fused upstream of the EGFP coding sequence (Fig. 1A). To generate founder fish, circular DNA constructs of Tol2GT2MP plasmid and the Tol2-transposase mRNA synthesized in vitro were coinjected into zebrafish embryos. The injected fish were raised to adulthood and mated. GFP-positive embryos were identified under a fluorescence microscope at 24-48 hours postfertilization (hpf). A total of 1,738 individual F1 fish lines were isolated and fluorescent protein reporter expression patterns of these lines exhibited a broad range of tissue specificities, including brain, spinal cord, floor plate, sensory organs, neural crest cells, notochord, blood vessels, gut, pancreas, skin, and primordial germ cells (data unpublished). To detect the insertion sites, a modified linker-mediated polymerase chain reaction (PCR) procedure based on previous reports32, 33 was performed. Genomic flanking sequences of the trap insertions were identified and cloned for sequencing. These sequences were used in BlastN searches in the zebrafish genomic database Ensembl (http://www.ensembl.org/). A sequence was regarded as a match to the zebrafish genome sequence only when they were over 95% identity.
Among these transgenic fish lines, a liver-specific screen was performed at later developmental stages (3 days postfertilization [dpf], 5 dpf). A total of 15 fish lines with liver-specific GFP expression were generated from the screen. Genomic flanking sequences were cloned and insertion sites were mapped in the zebrafish genome. One such line, mp255c, trapped the gene designated matrix metalloproteinase 23b (mmp23b, GeneID: 550589), which is orthologous to mammalian MMP23B (GeneID: 8510). Sequence analysis of the insertion site by linker-mediated PCR revealed that mmp23b was ≈6.1 kb downstream of the GFP insertion (Fig. 1B).
Mmp23b Is Specifically Expressed in the Developing Zebrafish Liver.
The enhancer trap line, mp255c, exhibits liver- and brain-specific GFP expression patterns (Fig. 1C,E). RNA WISH showed that mmp23b expression is mostly specific to the liver but has a minor domain in the brain (Fig. 1D,F). Overall, GFP expression in mp255c faithfully recapitulated the endogenous mmp23b expression in developing zebrafish embryos, including the liver and brain. Mmp23b was expressed throughout all developmental stages, starting at 1 dpf in the brain and peaking at 2-4 dpf after liver development began (Supporting Information Fig. S1C). Protein alignment and phylogenic tree analysis showed that zebrafish mmp23b shares significant homology with mouse MMP23 and human MMP23B (Supporting Information Fig. S1A,B). Reverse-transcription PCR (RT-PCR) analysis revealed that Mmp23 is also expressed in mouse liver (Supporting Information Fig. S1D) and brain.34 These studies indicate that mmp23b/Mmp23/MMP23B is highly conserved from zebrafish to human.
Knockdown of mmp23b Specifically Affects Liver Development.
To examine mmp23b function, we generated two MOs against mmp23b, blocking the translation start site (MO1) and the 5′ UTR (MO2), respectively. Injection of either MO caused no apparent morphological changes (Supporting Information Fig. S3B,F). Further analysis by WISH using a liver marker, ceruloplasmin(cp), revealed significant liver mass reduction in mmp23b morphants (Fig. 2A,B). We observed a similar reduction in liver GFP expression when the mmp23b:GFP line was injected with MO1 (Fig. 2C). To determine whether liver size reduction was tissue-specific, we analyzed gata6 expression, which is present in the liver, pancreas, and gut. Pancreatic and gut expression of gata6 in the mmp23b morphants was similar to that of control embryos (Fig. 2D), whereas the liver expression was significantly reduced (Fig. 2D, arrow). In addition, pancreatic expression of the endocrine cell marker insulin (ins) and exocrine cell marker carboxypeptidaseA (cpa) remained normal in mmp23b morphants (Supporting Information Fig. S2A,B). Furthermore, liver size reduction can be rescued with mmp23b mRNA coinjection with the MO (Supporting Information Fig. S4C).
To examine whether size reduction of the developing liver was due to a defect of progenitor cell specification, we performed WISH using a probe against hhex, one of the earliest liver markers in zebrafish. Endodermal expression of hhex at 36 hpf and 48 hpf (Fig. 2E,F) in zebrafish appears normal in the developing liver and pancreas of mmp23ab morphants. These results suggest that mmp23ab deficiency leads to an organ-specific size reduction in the developing liver that is independent of early hepatic specification.
Endothelial cells have been shown to be required for liver morphogenesis in a mouse model.35 The MMP family is also known to be involved in blood vessel formation and remodeling.36 We thus tested capillary morphogenesis in the zebrafish liver with an flk:RFP/mmp23b:GFP double transgenic fish line. However, there was no significant change of liver capillaries in mmp23b morphants at 3 dpf (Supporting Information Fig. S6).
Knockdown of mmp23b Causes Defect of Liver Cell Proliferation.
To investigate the underlying mechanism of liver size reduction in mmp23b morphants, we performed immunostaining analysis of pH3, a cell proliferation marker. In control embryos we detected an average of 11.3 ± 0.9 (n = 6) pH3-positive cells versus 0.8 ± 0.5 (n = 6, P < 0.0005) in mmp23b morphant livers (Fig. 3A-C). To assess whether cell death contributes to liver size reduction in mmp23b morphants, we conducted a Transferase-Mediated dUTP Nick-End Labeling (TUNEL) assay. Embryonic hepatocytes presented a low apoptotic index at 4 dpf, which was unchanged in mmp23b morphants (Fig. 3D,E). Therefore, reduction of liver size appears to be due to defective cell proliferation in the developing liver of mmp23b morphants.
Knockdown of tnfa or tnfb Induces Liver Phenotypes Similar to That of mmp23b Deficiency.
The proinflammatory cytokine TNF is a key regulator of liver homeostasis in mammals.37 There are two TNF isoforms in zebrafish, tnfa (GeneID: 405785) and tnfb (GeneID: 554167). To examine whether TNFs are required for liver development in zebrafish, we generated antisense MOs to knockdown tnfa or tnfb. Embryos injected with either tnfa MO or tnfb MO exhibited a severely underdeveloped liver at 3 dpf (Fig. 4A). In contrast, pancreas development was unaffected, as indicated by normal cpa expression in both tnfa or tnfb morphants (Fig. 4B). Therefore, tnfa and tnfb knockdown produces hepatic phenotypes similar to that of mmp23b morphants.
Forced Expression of tnfa or tnfb Restores Liver Development in mmp23b Morphants.
Because tnfa and tnfb morphants exhibit similarly underdeveloped livers as in mmp23b morphants, we tested whether mmp23b and TNF interact genetically. We analyzed expression levels of tnfa,tnfb, tnfr1a (tnfreceptor1a) in mmp23b morphants by quantitative RT-PCR. Knockdown of mmp23b decreased the expression level of both tnf ligands (tnfa 0.31 ± 0.05, P < 0.005; tnfb 0.10 ± 0.003, P < 0.0005) and receptor (tnfr1a 0.51 ± 0.07, P < 0.005; Fig. 4D). Then we coinjected mmp23b MO with tnfa or tnfb mRNA and analyzed liver development by cp WISH. At 5 dpf, injection of mmp23b MO alone resulted in a dramatically reduced liver size (Fig. 4C). However, coinjection of tnfa mRNA with mmp23b MO restored the liver size (Fig. 4C) comparable to that of control embryos. Coinjection of tnfb mRNA with mmp23b MO also rescued liver development (Fig. 4C), although to a lesser extent. Conversely, forced expression of mmp23b mRNA in tnfa or tnfb morphants failed to restore liver development (Supporting Information Fig. S4D-F). These data suggest that mmp23b acts upstream of tnf in liver development.
Mmp23b/MMP23B Is Involved in TNF Shedding in a Cell Culture System.
TNF is expressed on the cell surface as a transmembrane pro-protein and requires processing by TACE/ADAM17 to become active, although membrane-bound pro-TNF has some capacity as an inflammatory activator.15 The mmp23b gene encodes a 377 amino acids protein that contains a catalytic domain similar to that of TACE/ADAM17. This led us to postulate that Mmp23b is involved in TNF shedding. To test this hypothesis we took advantage of the null mutant TACE−/− fibroblasts, which has minimal endogenous TNF converting ability.15 In the control cells with cotransfection of HTNF-3xFLAG (human TNF) and CMV-GFP constructs, membrane-bound TNF (from cell lysates, 31 kD with FLAG,) and soluble TNF (from supernatants, 22 kD with FLAG) were detected by western blot using an anti-FLAG antibody (Fig. 5A). As expected, cotransfection of HMMP23B (human MMP23B) and TNF caused a significant increase of soluble TNF comparable to that of MTACE (mouse TACE) cotransfection (Fig. 5A). Western blots of secreted TNF and membrane-associated TNF showed both HMMP23B and MTACE could lead to an increase of soluble TNF. ELISA analysis with anti-HTNF was also performed separately to quantify the TNF release efficiency by HMMP23B (Fig. 5B). Compared to the control group, HMMP23B increased shedding of TNF by 2.32-fold, whereas MTACE increased by 2.36-fold (Fig. 5C). These data suggest that Mmp23b/MMP23B might be involved in TNF shedding.
To test if HMMP23B interacts with TNF, we performed an immunoprecipitation experiment. TACE−/− cells were transfected with CMV-MYC or HMMP23B-MYC, along with HTNF-3xFLAG. Protein lysates were immunoprecipitated with anti-MYC antibody and blotted with anti-FLAG antibody. We found that HMMP23B has a physical interaction with HTNF (Supporting Information Fig. S7), leading us to speculate that HMMP23B might be directly involved in the shedding process, either as a proteinase or as a cofactor.
Several MMPs have been previously implicated in liver injury in response to increased proinflammatory cytokines.5 However, none of them are expressed specifically in the liver under physiological conditions. Mmp23b is the first MMP member that mainly has liver-specific expression. In zebrafish it is initially expressed at 24 hpf and continues to be highly expressed in the developing liver (Fig. 1C-F; Supporting Information Fig. S1C). Preliminary analysis showed that the mammalian homolog of mmp23b is also expressed in normal liver cells (Supporting Information Fig. S1D), which is in contrast with other MMP family members activated primarily by liver injury and disease processes. Mmp23b is also the first MMP member that has been demonstrated to have a clear biological function in liver development. Knockdown of mmp23b resulted in a severe reduction of liver size without affecting early liver development marker hhex (Fig. 2E,F), suggesting that mmp23b is required for hepatocyte proliferation after initial hepatoblast determination and specification. The liver is the only organ in mammals capable of natural regeneration of lost tissue and as little as 25% of the liver can regenerate into a whole liver due to hepatocyte reenterance into the cell cycle. It is known that hepatocyte proliferation is controlled by the neurotrophin receptor p75NTR, which instructs HSCs to differentiate and then make growth factors and ECM to support hepatocyte proliferation.38 P75NTR is a TNF receptor superfamily member and it will be interesting to see if mmp23b is essential for liver cell proliferation during regeneration in response to p75NTR signaling.
There are 25 vertebrate MMPs including two subfamilies, membrane-bound and nonmembrane-bound.1 Mmp23b/MMP23B contains a type II transmembrane domain at the amino-terminal that likely enables it membrane-bounded. Due to the unavailability of a zebrafish hepatocyte in vitro cell culture system, we took advantage of the broadly used TACE−/− fibroblast cell line system. We provide evidence that Mmp23b/MMP23B is also involved in TNF shedding using the cell culture system, suggesting membrane-bound MMP family members can process cytokines in a tissue-specific manner. Epistatic studies in zebrafish place TNF downstream of mmp23b in liver development, which is consistent with biochemical data showing that TNFs are related to Mmp23b/MMP23B activity. Although our in vitro immunoprecipitation experiment suggests that there are direct interactions between HMMP23B and HTNF, HMMP23B's in vivo biochemical partners in regulating TNF releasing remains to be determined. Previous immunoprecipitation and western blotting analysis have also shown that TACE could interact with the proform of TNF.39 It is likely that other MMPs including MMP23A and closely related MMP19 may share a similar specificity. Further study about the mechanism of the interaction is necessary to understand the roles of MMPs in TNF activation.
The MMP family is involved in blood vessel formation and remodeling, which is necessary for liver morphogenesis in mice. In this study, no capillaries appear affected by mmp23b knockdown. Therefore, the function of mmp23b in liver proliferation is independent of capillary morphogenesis. This finding is consistent with previous studies in zebrafish because liver and biliary tree form normally in the absence of blood vessels in cloche mutants.40, 41
Like other MMPs, mmp23b may have more than one target contributing to inhibited cell proliferation. Although we show here that MMP23B interacts with TNFs, it may have other cytokine targets such as transforming growth factor-β1 (TGF-β1), which is also an important indicator of liver fibrosis.42 TGF-β1 is released by cells but with its cleaved prodomain still latently associated. Several mechanisms, including MMP proteolysis (MMP3, MMP9, and MMP14), have been proposed to release the active cytokine from this complex.8 It is possible that MMP23B can also act in the TGF-β1 releasing process in vivo. Recently, Wyatt et al.21 developed two novel assays that allow the detection and characterization of active MMPs in vivo (differential in vivo zymography and activity-based protease profiling). These techniques should be useful for further detection of in vivo MMP23B activities and its potential substrates. Activity-based protease profiling allows detection of biologically activity of MMP23B during embryonic development. Fluorogenic TNF or TGF-β1 as well as some other candidates could be injected into a living zebrafish embryo, and then visualized for the fluorescent breakdown products by confocal microscopy. The fluorescent breakdown products of observed in liver could be a candidate substrate of MMP23B.
Hepatic fibrosis represents the final common pathway of chronic injuries associated with viral hepatitis, alcohol abuse, autoimmune diseases, drugs, and metabolic syndromes.43 In nonalcoholic fatty liver disease, the disease spectrum ranges from early hepatic steatosis to endstage liver disease, with fibrosis and cirrhosis accounting for the most common cause of abnormal liver function tests and an important cause of liver transplants in the United States. Several MMP family members, which normally have relatively low expression in the liver, have been shown to be abnormally activated in fibrosis.4 On the other hand, mmp23b is highly expressed in normal livers, appearing to be different from those MMP family members involved in fibrosis. We did some preliminary experiments to investigate whether partial mmp23b deficiency might be related to liver fibrosis. Several markers of gene expression for fibrosis were changed in a way that reflects the ontogeny of fibrosis, as detected by quantitative RT-PCR (Supporting Information Fig. S8). These findings, although preliminary, suggest that deficiency of mmp23b is likely a marker or a cause of early liver fibrosis. However, further studies, including the generation of mmp23b genetic mutations, are needed to link this gene to liver fibrosis. We failed to detect collagen deposition by trichrome staining in mmp23b-deficient zebrafish embryos at 6 dpf, possibly due to the short half-life of antisense MO. Mmp23b is also unique in relation to liver carcinogenesis as it has been shown by gene expression profiling of zebrafish liver cancer samples that mmp23b is significantly decreased, whereas other MMPs are increased.23 Again, it will be interesting to determine if mmp23b/MMP23B deficiency represents one of the causes leading to liver cancer, whereas the increases of other MMPs are a consequence of hepatic carcinogenesis.
We thank numerous members of the S. Lin and B. Zhang laboratories for excellent fish care, technical assistance, and helpful discussions; R. Black for sharing TACE−/− cell line; J. Kolls for sharing HTNF-3xFLAG plasmid.