Mol. Cells 2020; 43(11): 945-952
Published online November 18, 2020
https://doi.org/10.14348/molcells.2020.0100
© The Korean Society for Molecular and Cellular Biology
Correspondence to : hspark@uos.ac.kr
Hypoxia induces the expression of several genes through the activation of a master transcription factor, hypoxia-inducible factor (HIF)-1α. This study shows that hypoxia strongly induced the expression of two carboxypeptidases (CP), CPA4 and CPE, in an HIF-1α-dependent manner. The hypoxic induction of CPA4 and CPE gene was accompanied by the recruitment of HIF-1α and upregulation in the active histone modification, H3K4me3, at their promoter regions. The hypoxic responsiveness of CPA4 and CPE genes was observed in human adipocytes, human adipose-derived stem cells, and human primary fibroblasts but not mouse primary adipocyte progenitor cells. CPA4 and CPE have been identified as secreted exopeptidases that degrade and process other secreted proteins and matrix proteins. This finding suggests that hypoxia changes the microenvironment of the obese hypoxic adipose tissue by inducing the expression of not only adipokines but also peptidases such as CPA4 and CPE.
Keywords carboxypeptidase A4, carboxypeptidase E, human adipose-derived stem cells, hypoxia, hypoxia-inducible factor-1α
Hypoxic microenvironment has been associated with cancer progression and stemness maintenance. Hypoxia changes the expression of several target genes, but the mechanism underlying the hypoxia-mediated pleiotropic effects on metabolic status, cell cycle, differentiation, angiogenesis, cell migration, and metastasis is unclear (Moon et al., 2015). Hypoxia-inducible factor (HIF) is a master transcription factor comprising HIF-α and HIF-1β (also known as aryl hydrocarbon nuclear translocator, ARNT) subunits. HIF-α subunit is sensitive to changes in O2 concentration because its stability and trans-activity are regulated by two different HIF-α-specific O2 and a-ketoglutarate-dependent dioxygenases, namely, prolyl hydroxylases (PHD) and asparaginyl hydroxylase (also known as factor-inhibiting HIF, FIH). HIF-1α is hydroxylated at proline 564 and/or 402 residues by PHD using O2. The hydroxylated proline residues of HIF-1α are recognized by an E3 ubiquitin ligase, von Hippel Lindau protein, that initiates the ubiquitin-dependent degradation of HIF-1α. FIH hydroxylates the asparagine 803 residue of HIF-1α, and prevents the binding of the coactivators CBP/p300 to the C-terminal region of HIF-1α. These two enzymes are inactivated under hypoxia, leading to the stabilization of HIF-1α and its subsequent translocation into the nucleus to form a heterodimer with HIF-1β. HIF-1 heterodimer interacts with E-box-like hypoxia-responsive element to induce the expression of target genes (Gorlach et al., 2015).
Hypoxic regions in human body are often found where O2 consumption exceeds O2 supply. HIF-1α is activated at the sight of inflammation because inflammatory processes increase O2 consumption owing to an increase in oxidation reactions catalyzed by NADPH oxidases and an increase in the production of reactive oxygen species, which are inhibitors of PHD and FIH (Lee et al., 2011). O2 supply is limited in avascular regions such as the inner core of solid tumors, stem cell niche, and the obese adipose tissue. In the obese adipose tissue, hypertrophic adipocytes move further away from blood vessels and become hypoxic. Adipocytes have been re-evaluated for their endocrine functions because they secreted many cytokines and hormones such as inflammatory cytokines, leptin, and adiponectin (Makki et al., 2013). These secreted proteins from adipocytes are termed as adipokines. In comparison with lean adipocytes, obese adipocytes upregulate the expression and secretion of many inflammatory adipokines such as interleukin-6, leptin, and plasminogen activator inhibitor-1, most of which are target genes of HIF-1α (Lee et al., 2014). Beside adipocytes, the adipose tissue is made of other cells such as endothelial cells, macrophages, and mesenchymal stem cells. In our previous microarray analyses, we identified several novel target genes that are upregulated by hypoxia in human adipose-derived stem cells (hADSCs) (Lee et al., 2017). We first found that the expression of carboxypeptidase A4 (CPA4) and carboxypeptidase E (CPE) was highly upregulated in hypoxic hADSCs (Lee et al., 2017). The hypoxic environment of hypertrophic adipocytes increases the expression of CPA4 and CPE, which function by processing other secreted proteins and matrix proteins in the adipose tissue. This finding indicates that hypoxia changes the microenvironment of the obese adipose tissue by inducing the expression of not only adipokines but also peptidases such as CPA4 and CPE from adipose-derived mesenchymal stem cells.
Anti-HIF-1α (610958) and anti-CPE (610758) antibodies were purchased from BD Biosciences (USA). Anti-CPA4 (ab81543) and anti-14-3-3γ (MAB5700) antibodies were obtained from Abcam (USA) and R&D Systems (USA), respectively. Mouse IgG (sc-2025) and anti-H3K4me3 (9751S) were obtained from Santa Cruz Biotechnology (USA) and Cell Signaling Technology (USA), respectively. Ponceau S solution and Oil Red O stain were supplied by Sigma-Aldrich (USA).
hADSCs (catalog No. 510070, lot No. 2033) were purchased from Invitrogen (USA) and maintained as previously described (Lee et al., 2017). IMR90 human fetal lung fibroblasts were obtained from the American Type Culture Collection (ATCC, USA) and maintained in an Eagle’s minimum essential medium (EMEM) supplemented with 10% (v/v) fetal bovine serum. Cells were exposed to hypoxia through incubation in a Forma anaerobic incubator (model 1029; Thermo Fisher Scientific, USA) in an atmosphere of 5% CO2, 95% N2, and 0.5% O2. HIF-1α expression was knocked down in hADSCs using a pLKO.1 lentiviral vector (Addgene, USA) encoding either control a short-hairpin RNA (shRNA) or a shRNA sequence against human HIF-1α (5′-CCA GTT ATG ATT GTG AAG TTA-3′). For adipocyte differentiation, hADSCs were cultured in an adipogenic medium (catalog No. A1007001; Invitrogen); the medium was replaced every 3 days for 12 days according to the manufacturer’s instructions. Mouse adipocyte progenitor cells were isolated from the subvascular fraction (SVF) of the subcutaneous white adipose tissue (sWAT) of an 8-week-old C57BL/6J strain using an adipose tissue progenitor isolation kit (Miltenyi Biotec, Germany) according to the manufacturer’s instructions. The SVF was obtained after collagenase digestion of the sWAT using a previously described method (Freshney, 2000). Conditioned medium (CM) was a spent medium harvested from hADSCs cultured under normoxia (21% O2) or hypoxia (0.5% O2) for 2 days (Park et al., 2013). CM was centrifuged to remove debris and its supernatant was used.
Steady-state RNA expression was measured by real-time qPCR using Power SYBR Green PCR master mix on a QuantStudio 3 real-time PCR system (Applied Biosystems, USA). The Ct value of target mRNA was normalized against that of endogenous 18S rRNA (ΔCt = Ct target – Ct 18S). Ct is the threshold cycle of qPCR defined by the QuantStudio 3 real-time PCR system. The relative mRNA level of a target gene is obtained by 2-ΔΔCt; ΔΔCt = ΔCt treated value – ΔCt untreated value. Pairs of forward and reverse primer sequences for RT-qPCR were as follows: human
ChIP analysis was performed as previously described (Lee et al., 2017). The Ct value of target gene from the immunoprecipitated DNA sample was normalized against the Ct value of target gene in input DNA (Δ
All quantitative measurements were performed through at least two independent experiments. For comparison between two groups,
The microarray data of hypoxic hADSCs revealed the hypoxia-mediated increase in the mRNA expression levels of several carboxypeptidases (Lee et al., 2017). Among the 21 carboxypeptidases,
We exposed IMR90 human fetal lung fibroblasts to hypoxia to investigate whether hypoxia induces the expression of CPA4 and CPE in other primary human cells. In comparison with IMR90 fibroblasts, hADSCs were more sensitive to hypoxia in terms of induction of
We also tested whether hADSCs maintain their responsiveness to hypoxia for the induction of
To test whether HIF-1α is essential for mediating the hypoxia-induced expression of
This study shows that hypoxic condition dramatically induced the expression of two carboxypeptidases CPA4 and CPE in an HIF-1α-dependent manner. The hypoxic responsiveness of
Considering that hypoxic regions are often found in the obese adipose tissue, our findings suggest the possibility that hypoxia-mediated induction of CPA4 and CPE expression in the obese adipose tissue may contribute to the upregulation in secreted CPA4 and CPE proteins in the serum, ultimately leading to increased tumor metastasis. Hypoxia is an important environmental condition that determines cell fate by reconstituting microenvironment through induction of target secretory proteins. This study suggests that hypoxic induction of secretory CPE and CPA4 proteins in hADSCs further changes the extracellular microenvironment by processing diverse extracellular substrates of CPE and CPA4.
This work was supported by the 2018 sabbatical year research grant of the University of Seoul for H Park.
Y.M., R.M., and H.P. conceived ideas, performed experiments and wrote the manuscript. H.R., S.C., and S.L. performed experiments.
The authors have no potential conflicts of interest to disclose.
Mol. Cells 2020; 43(11): 945-952
Published online November 30, 2020 https://doi.org/10.14348/molcells.2020.0100
Copyright © The Korean Society for Molecular and Cellular Biology.
Yunwon Moon1,2,3 , Ramhee Moon1,3
, Hyunsoo Roh1
, Soojeong Chang1
, Seongyeol Lee1
, and Hyunsung Park1,*
1Department of Life Science, University of Seoul, Seoul 02504, Korea, 2Present address: Cell Therapy Research Center, GC LabCell, Yongin 16924, Korea, 3These authors contributed equally to this work.
Correspondence to:hspark@uos.ac.kr
Hypoxia induces the expression of several genes through the activation of a master transcription factor, hypoxia-inducible factor (HIF)-1α. This study shows that hypoxia strongly induced the expression of two carboxypeptidases (CP), CPA4 and CPE, in an HIF-1α-dependent manner. The hypoxic induction of CPA4 and CPE gene was accompanied by the recruitment of HIF-1α and upregulation in the active histone modification, H3K4me3, at their promoter regions. The hypoxic responsiveness of CPA4 and CPE genes was observed in human adipocytes, human adipose-derived stem cells, and human primary fibroblasts but not mouse primary adipocyte progenitor cells. CPA4 and CPE have been identified as secreted exopeptidases that degrade and process other secreted proteins and matrix proteins. This finding suggests that hypoxia changes the microenvironment of the obese hypoxic adipose tissue by inducing the expression of not only adipokines but also peptidases such as CPA4 and CPE.
Keywords: carboxypeptidase A4, carboxypeptidase E, human adipose-derived stem cells, hypoxia, hypoxia-inducible factor-1α
Hypoxic microenvironment has been associated with cancer progression and stemness maintenance. Hypoxia changes the expression of several target genes, but the mechanism underlying the hypoxia-mediated pleiotropic effects on metabolic status, cell cycle, differentiation, angiogenesis, cell migration, and metastasis is unclear (Moon et al., 2015). Hypoxia-inducible factor (HIF) is a master transcription factor comprising HIF-α and HIF-1β (also known as aryl hydrocarbon nuclear translocator, ARNT) subunits. HIF-α subunit is sensitive to changes in O2 concentration because its stability and trans-activity are regulated by two different HIF-α-specific O2 and a-ketoglutarate-dependent dioxygenases, namely, prolyl hydroxylases (PHD) and asparaginyl hydroxylase (also known as factor-inhibiting HIF, FIH). HIF-1α is hydroxylated at proline 564 and/or 402 residues by PHD using O2. The hydroxylated proline residues of HIF-1α are recognized by an E3 ubiquitin ligase, von Hippel Lindau protein, that initiates the ubiquitin-dependent degradation of HIF-1α. FIH hydroxylates the asparagine 803 residue of HIF-1α, and prevents the binding of the coactivators CBP/p300 to the C-terminal region of HIF-1α. These two enzymes are inactivated under hypoxia, leading to the stabilization of HIF-1α and its subsequent translocation into the nucleus to form a heterodimer with HIF-1β. HIF-1 heterodimer interacts with E-box-like hypoxia-responsive element to induce the expression of target genes (Gorlach et al., 2015).
Hypoxic regions in human body are often found where O2 consumption exceeds O2 supply. HIF-1α is activated at the sight of inflammation because inflammatory processes increase O2 consumption owing to an increase in oxidation reactions catalyzed by NADPH oxidases and an increase in the production of reactive oxygen species, which are inhibitors of PHD and FIH (Lee et al., 2011). O2 supply is limited in avascular regions such as the inner core of solid tumors, stem cell niche, and the obese adipose tissue. In the obese adipose tissue, hypertrophic adipocytes move further away from blood vessels and become hypoxic. Adipocytes have been re-evaluated for their endocrine functions because they secreted many cytokines and hormones such as inflammatory cytokines, leptin, and adiponectin (Makki et al., 2013). These secreted proteins from adipocytes are termed as adipokines. In comparison with lean adipocytes, obese adipocytes upregulate the expression and secretion of many inflammatory adipokines such as interleukin-6, leptin, and plasminogen activator inhibitor-1, most of which are target genes of HIF-1α (Lee et al., 2014). Beside adipocytes, the adipose tissue is made of other cells such as endothelial cells, macrophages, and mesenchymal stem cells. In our previous microarray analyses, we identified several novel target genes that are upregulated by hypoxia in human adipose-derived stem cells (hADSCs) (Lee et al., 2017). We first found that the expression of carboxypeptidase A4 (CPA4) and carboxypeptidase E (CPE) was highly upregulated in hypoxic hADSCs (Lee et al., 2017). The hypoxic environment of hypertrophic adipocytes increases the expression of CPA4 and CPE, which function by processing other secreted proteins and matrix proteins in the adipose tissue. This finding indicates that hypoxia changes the microenvironment of the obese adipose tissue by inducing the expression of not only adipokines but also peptidases such as CPA4 and CPE from adipose-derived mesenchymal stem cells.
Anti-HIF-1α (610958) and anti-CPE (610758) antibodies were purchased from BD Biosciences (USA). Anti-CPA4 (ab81543) and anti-14-3-3γ (MAB5700) antibodies were obtained from Abcam (USA) and R&D Systems (USA), respectively. Mouse IgG (sc-2025) and anti-H3K4me3 (9751S) were obtained from Santa Cruz Biotechnology (USA) and Cell Signaling Technology (USA), respectively. Ponceau S solution and Oil Red O stain were supplied by Sigma-Aldrich (USA).
hADSCs (catalog No. 510070, lot No. 2033) were purchased from Invitrogen (USA) and maintained as previously described (Lee et al., 2017). IMR90 human fetal lung fibroblasts were obtained from the American Type Culture Collection (ATCC, USA) and maintained in an Eagle’s minimum essential medium (EMEM) supplemented with 10% (v/v) fetal bovine serum. Cells were exposed to hypoxia through incubation in a Forma anaerobic incubator (model 1029; Thermo Fisher Scientific, USA) in an atmosphere of 5% CO2, 95% N2, and 0.5% O2. HIF-1α expression was knocked down in hADSCs using a pLKO.1 lentiviral vector (Addgene, USA) encoding either control a short-hairpin RNA (shRNA) or a shRNA sequence against human HIF-1α (5′-CCA GTT ATG ATT GTG AAG TTA-3′). For adipocyte differentiation, hADSCs were cultured in an adipogenic medium (catalog No. A1007001; Invitrogen); the medium was replaced every 3 days for 12 days according to the manufacturer’s instructions. Mouse adipocyte progenitor cells were isolated from the subvascular fraction (SVF) of the subcutaneous white adipose tissue (sWAT) of an 8-week-old C57BL/6J strain using an adipose tissue progenitor isolation kit (Miltenyi Biotec, Germany) according to the manufacturer’s instructions. The SVF was obtained after collagenase digestion of the sWAT using a previously described method (Freshney, 2000). Conditioned medium (CM) was a spent medium harvested from hADSCs cultured under normoxia (21% O2) or hypoxia (0.5% O2) for 2 days (Park et al., 2013). CM was centrifuged to remove debris and its supernatant was used.
Steady-state RNA expression was measured by real-time qPCR using Power SYBR Green PCR master mix on a QuantStudio 3 real-time PCR system (Applied Biosystems, USA). The Ct value of target mRNA was normalized against that of endogenous 18S rRNA (ΔCt = Ct target – Ct 18S). Ct is the threshold cycle of qPCR defined by the QuantStudio 3 real-time PCR system. The relative mRNA level of a target gene is obtained by 2-ΔΔCt; ΔΔCt = ΔCt treated value – ΔCt untreated value. Pairs of forward and reverse primer sequences for RT-qPCR were as follows: human
ChIP analysis was performed as previously described (Lee et al., 2017). The Ct value of target gene from the immunoprecipitated DNA sample was normalized against the Ct value of target gene in input DNA (Δ
All quantitative measurements were performed through at least two independent experiments. For comparison between two groups,
The microarray data of hypoxic hADSCs revealed the hypoxia-mediated increase in the mRNA expression levels of several carboxypeptidases (Lee et al., 2017). Among the 21 carboxypeptidases,
We exposed IMR90 human fetal lung fibroblasts to hypoxia to investigate whether hypoxia induces the expression of CPA4 and CPE in other primary human cells. In comparison with IMR90 fibroblasts, hADSCs were more sensitive to hypoxia in terms of induction of
We also tested whether hADSCs maintain their responsiveness to hypoxia for the induction of
To test whether HIF-1α is essential for mediating the hypoxia-induced expression of
This study shows that hypoxic condition dramatically induced the expression of two carboxypeptidases CPA4 and CPE in an HIF-1α-dependent manner. The hypoxic responsiveness of
Considering that hypoxic regions are often found in the obese adipose tissue, our findings suggest the possibility that hypoxia-mediated induction of CPA4 and CPE expression in the obese adipose tissue may contribute to the upregulation in secreted CPA4 and CPE proteins in the serum, ultimately leading to increased tumor metastasis. Hypoxia is an important environmental condition that determines cell fate by reconstituting microenvironment through induction of target secretory proteins. This study suggests that hypoxic induction of secretory CPE and CPA4 proteins in hADSCs further changes the extracellular microenvironment by processing diverse extracellular substrates of CPE and CPA4.
This work was supported by the 2018 sabbatical year research grant of the University of Seoul for H Park.
Y.M., R.M., and H.P. conceived ideas, performed experiments and wrote the manuscript. H.R., S.C., and S.L. performed experiments.
The authors have no potential conflicts of interest to disclose.
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