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. 2016 Apr 13;30(6):587–599. doi: 10.1210/me.2015-1302

TIMP3 Modulates GHR Abundance and GH Sensitivity

Yue Zhang 1, Xiangdong Wang 1, Kimberly Loesch 1, Larry A May 1, George E Davis 1, Jing Jiang 1, Stuart J Frank 1,✉
PMCID: PMC4884343  PMID: 27075707

Abstract

GH receptor (GHR) binds GH at the cell surface via its extracellular domain and initiates intracellular signal transduction, resulting in important anabolic and metabolic actions. GH signaling is subject to dynamic regulation, which in part is exerted by modulation of cell surface GHR levels. Constitutive and inducible metalloprotease-mediated cleavage of GHR regulate GHR abundance and thereby modulate GH action. We previously demonstrated that GHR proteolysis is catalyzed by the TNF-α converting enzyme (TACE; ADAM17). Tissue inhibitors of metalloproteases-3 (TIMP3) is a natural specific inhibitor of TACE, although mechanisms underlying this inhibition are not yet fully understood. In the current study, we use two model cell lines to examine the relationships between cellular TACE, TIMP3 expression, GHR metalloproteolysis, and GH sensitivity. These two cell lines exhibited markedly different sensitivity to inducible GHR proteolysis, which correlated directly to their relative levels of mature TACE vs unprocessed TACE precursor and indirectly to their levels of cellular TIMP3. Our results implicate TIMP3 as a modulator of cell surface GHR abundance and the ability of GH to promote cellular signaling; these modulatory effects may be conferred by endogenous TIMP3 expression as well as exogenous TIMP3 exposure. Furthermore, our analysis suggests that TIMP3, in addition to regulating the activity of TACE, may also modulate the maturation of TACE, thereby affecting the abundance of the active form of the enzyme.

GH is a 22-kDa polypeptide vertebrate hormone derived largely from the anterior pituitary gland. GH signals powerful somatogenic and metabolic effects and may have a role in cancer cell behavior (1–4) by interacting with the cell surface GH receptor (GHR), a 620-residue transmembrane glycoprotein member of the cytokine receptor superfamily (5–7). GH-induced GHR binding causes activation of the GHR-associated tyrosine kinase, Janus kinase (JAK)-2, and downstream signaling pathways, including the signal transducer and activator of transcription (STAT)-5 pathway (8–10).

Cell surface GHR abundance is a key determinant of GH sensitivity that is regulated by cells by several means (11–13). Among these mechanisms, GHR is targeted for constitutive and inducible metalloprotease-mediated cleavage that alters surface GHR levels and can modulate GH signaling. GHR metalloproteolysis occurs in the proximal extracellular domain stem region and results in loss of the full-length receptor, appearance of a cell-associated cytoplasmic domain-containing GHR fragment (the remnant protein), and shedding of a soluble GHR extracellular domain (the GH binding protein [GHBP]) (14–16). In cultured cells, GHR proteolysis can be induced by serum, platelet-derived growth factor, or phorbol-12-myristate-12-acetate (PMA) and results in the desensitization to subsequent GH treatment (17, 18). Similarly, treatment of mice with endotoxin also elicits this proteolytic GHR down-regulation and desensitization to hepatic GH action (19).

We previously demonstrated that GHR proteolysis is catalyzed by the TNF-α converting enzyme (TACE; ADAM17) (17), a disintegrin and metalloprotease (ADAM) family member first described as the enzyme responsible for the generation of the soluble TNF-α through the cleavage of its membrane-bound precursor (20, 21). TACE is noteworthy in that it is a transmembrane protein with a zinc-dependent catalytic region residing in its extracellular domain. In addition to TNF-α and GHR, TACE cleaves numerous transmembrane substrates, including amyloid precursor protein, Notch1, and epidermal growth factor family ligands, in their extracellular domains (22–24). Tissue inhibitors of metalloproteases (TIMP) are soluble proteins that regulate metalloprotease activity by noncovalent interaction with the protease (25). TIMP3 is unique among family members because it is a physiologically relevant specific inhibitor of TACE (26, 27), although the mechanism of TIMP3's inhibition of TACE is incompletely known.

Our prior work established that inducible TACE-mediated proteolysis of GHR is a determinant of cellular GH sensitivity and that pretreatment with a hydroxamate-based chemical TACE inhibitor prevented inducible GHR proteolysis and rendered cells resistant to TACE-mediated GH desensitization (18). In the current study, we examine the relationships between cellular TIMP3 expression, GHR metalloproteolysis, and GH sensitivity. Our results implicate TIMP3 as a modulator of cell surface GHR abundance and the ability of GH to promote cellular signaling; this effect on GHR availability may be conferred by endogenous TIMP3 expression, as well as exogenous TIMP3 exposure. Furthermore, our analysis suggests that TIMP3, in addition to regulating the activity of TACE, may also modulate the maturation of TACE, thereby affecting the abundance of the active form of the enzyme.

Materials and Methods

Materials

Routine reagents were purchased from Sigma-Aldrich Corp unless otherwise noted. Fetal bovine serum, gentamicin sulfate, penicillin, and streptomycin were purchased from BioFluids. Recombinant human GH was kindly provided by Eli Lilly & Co.

Antibodies

Polyclonal anti-phospho-STAT5 was purchased from Cell Signaling Technology, Inc. Polyclonal anti-STAT5 was purchased from Santa Cruz Biotechnology. Monoclonal antiphosphotyrosine (pY, 4G10) was purchased from Upstate Biotechnology, Inc. Polyclonal anti-TIMP2 and anti-TIMP3 were purchased from EMD Millipore Corp. Polyclonal anti-GHRcytAL-47 (referred to as anti-GHR) against the intracellular domain of GHR (28), anti-JAK2AL33 (referred to as anti-JAK2) (28), anti-TACEAL45 (referred to as anti-TACE) (29), and monoclonal anti-GHRcyt-mAb against the intercellular domain of GHR (30) were described previously.

Cell culture and stable transfection

C14 cells were established and maintained as described previously (31). To obtain human embryonic kidney 293 (HEK293)-GHR-JAK2 cells, transfection of HEK293-GHR cells (32) (generously provided by Dr. Richard J. Ross, University of Sheffield, Sheffield, United Kingdom) with an expression vector encoding mouse JAK2 was performed using Lipofectamine Plus (Invitrogen Life Technologies, Inc) according to the manufacturer's instructions. Stably transfected clones were selected by growth on the G418-containing medium. HEK293-GHR-JAK2 cells were maintained in DMEM (1 g/L glucose) (Mediatech) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, 200 μg/mL G418, and 200 μg/mL zeocin.

Adenovirus preparation and infection

Adenoviruses (Ad) driving expression of TIMP2 and TIMP3 (Ad-TIMP2 and Ad-TIMP3) (33) were amplified and purified, as previously described (34). Concentration of purified virus was calculated by measuring the value of OD260. Cells were cultured to 70% confluence and infected with adenovirus at the indicated multiplicity of infection (MOI) in the absence of serum. After 1 hour, complete culture medium containing 10% fetal bovine serum was added, and the cells were allowed to recover for 48 hours before stimulation.

Cell starvation, cell stimulation, and protein extraction

Serum starvation of cells was accomplished by substitution of 0.5% (wt/vol) bovine serum albumin (fraction V: Roche Molecular Biochemicals) for fetal bovine serum in the culture medium for 16–20 hours prior to experiments. Pretreatments and stimulations were carried out at 37ºC in serum-free medium. Stimulations were terminated by washing the cells once with ice-cold PBS supplemented with 0.4 mM sodium orthovanadate and then harvested in lysis buffer (1% Triton X-100, 150 mM NaCL, 10% glycerol, 50 mM Tris-HCL, 100 mM NaF, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 20 mM 1,10 Phenanthroline, 1 mM sodium orthovanadate, 10 mM benzamidine, 5 μg/mL aprotinin, and 5 μg/mL leupeptin). Cells were lysed for 30 minutes at 4ºC in lysis buffer before centrifugation at 15 000 × g for 10 minutes at 4ºC. The protein concentration was determined and equal aliquots of protein extracts (supernatant) were subjected to immunoprecipitation or were directly electrophoresed and immunoblotted as indicated.

RNA interference (small interfering RNA [siRNA])

Both TIMP3 Silencer Predesigned siRNA and Silencer negative control 3 siRNA was purchased from Ambion, Inc. HEK293-GHR-JAK2 cells were plated transfected with siRNA using Thermo Scientific DharmaFECT transfection reagents (Thermo Fisher Scientific, Inc) following the manufacture's protocol. One day after siRNA transfection, transfected cells were split. Twenty-four hours later, cells were serum starved for another 16–20 hours. Three days after transfection, the cells were treated with GH, washed, and harvested in lysis buffer. Cell extracts were either subjected to immunoprecipitation to evaluate TIMP3 knockdown and GHR abundance or directly electrophoresed and immunoblotted to examine STAT5 activation.

Immunoprecipitation, enzymatic deglycosylation, and Western blotting

For immunoprecipitation, 0.5–1 mg protein was incubated with antibody against JAK2, GHR, TACE, or TIMP3 overnight at 4ºC. Protein A (or G)-agarose (fast flow; Pharmacia Biotech) was then added, and incubations continued for 1 hour at 4ºC. The beads were washed five times with lysis buffer. Sodium dodecyl sulfate (SDS) sample buffer eluates were resolved by SDS-PAGE and immunoblotted.

For enzymatic deglycosylation experiments, immunoprecipitation was first performed as above. Precipitated proteins were eluted by boiling the protein A (or G)-Sepharose beads in 0.5% SDS and 3% 2-mercaptoethanol for 5 minutes. Deglycosylation in 125 μL was accomplished by adjusting the buffer to include the following: 50 mm NaOAc, pH 5.5; 50 mm EDTA; 0.4% (vol/vol) Nonidet P-40; 6.4 mm phenylmethylsulfonyl fluoride; 0.1% SDS; 0.6% 2-mercaptoethanol; and 0.3 U of endoglycosidase F/neuraminidase-glycosidase F (Roche Molecular Biochemicals) at 37ºC for 16 hours. Nondeglycosylated controls were subjected to the same treatment, but without the addition of the glycosidase mixture. After the addition of SDS sample buffer, the proteins were subjected to SDS-PAGE and immunoblotting as indicated.

Proteins were resolved under reduced conditions by SDS-PAGE, and transferred to nitrocellulose membrane (Amersham Biosciences), followed by blocking with 2% BSA. Western transfers were immunoblotted with anti-pY (4G10) (1:2000), anti-JAK2AL33 (1:1000), anti-GHRcyt-AL-47 (1:1000), anti-TIMP2 (1:1000), anti-TIMP3 (1:1000), or anti-TACEAL45 (1:1000) antibodies.

Densitometric analysis

Densitometric quantitation of the immunoblots was performed using the ImageJ 1.30 program (developed by W. S. Rasband, Research Services Branch, National Institutes of Health, Bethesda, Maryland). Pooled data from several experiments are displayed as mean ± SE. The maximum signal was considered as 100% (except in Figures 3 and 4). The significance of differences (P value) of pooled results was estimated using unpaired t tests.

Figure 3.

Figure 3.

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Ratio of steady-state mature TACE to pro-TACE abundance is greater in C14 Cells compared with HEK293-GHR-JAK2 cells. A, C14 cells or HEK293-GHR-JAK2 cells were serum starved and detergent extracts were immunoprecipitated with anti-TACEAL45 or nonimmune serum as control (NI). Precipitates were divided into three portions and treated with endoH (Endo H), its vehicle control (−), or a combination of N-glycosidase F/N. Eluates of the precipitates from each treatment were resolved by SDS-PAGE and immunoblotted with anti-TACE. The immunoblots shown are representative of three such experiments. B, Densitometric analysis of data from three independent experiments, including that presented in panel A. Data are expressed as mean ± SE. C, Detergent extracts of serum-starved C14 cells or HEK293-GHR-JAK2 cells were immunoprecipitated with anti-TIMP3 or nonimmune serum as control (NI). Eluates were separated by SDS-PAGE and immunoblotted with anti-TIMP3. IP, immunoprecipitation; WB, Western blot.

Figure 4.

Figure 4.

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Forced TIMP3 expression in C14 cells reduces the mature TACE to pro-TACE ratio. A, C14 cells were infected with Ad-GFP, Ad-TIMP2, or Ad-TIMP3 at 400 MOI. Two days later, serum-starved cells were washed with cold PBS and harvested in lysis buffer. Detergent cell extracts were immunoprecipitated with anti-TACE. Eluates were separated by SDS-PAGE and immunoblotted with anti-TACE (upper panel). Detergent extracts were resolved by SDS-PAGE and immunoblotted with anti-TIMP3 (lower panel). The immunoblots shown are representative of three such experiments. B, Densitometric analysis of data from three separate experiments, including that presented in the upper panel of panel A. Data are expressed as mean ± SE. IP, immunoprecipitation; WB, Western blot.

Results

A GH-responsive HEK293 cell line that expresses GHR and JAK2

We previously studied the process and GH signaling impact of TACE-mediated GHR proteolysis in a human cell line, C14, in which GHR and JAK2 are stably expressed in the GHR- and JAK2-deficient human fibrosarcoma cell line, γ2A (35–40). To complement this system, we similarly created for the current study another human cell transfectant, HEK293-GHR-JAK2, in which JAK2 and human GHR are stably expressed in HEK293 cells. To verify GH responsiveness, HEK293-GHR-JAK2 cells were treated with vehicle or GH (500 ng/mL) for 10 minutes, after which detergent extracts were immunoprecipitated with anti-GHR monoclonal antibody vs a negative control (NI) (Figure 1A) or anti-JAK2 serum (Figure 1B). Eluates were subjected to SDS-PAGE and anti-GHR, anti-JAK2, and anti-pY blotting, demonstrating GH-induced tyrosine phosphorylation of the mature form of the GHR and of JAK2 in these cells. Consistent with GH-induced JAK2 activation and GHR phosphorylation, STAT5 phosphorylation was also detected in response to acute GH stimulation of these cells (Figure 1C). Thus, HEK293-GHR-JAK2 is a biochemically tractable GH-responsive cell system.

Figure 1.

Figure 1.

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GH activates GHR and the JAK2/STAT5 pathway in the human embryonic kidney cell line, HEK293-GHR-JAK2 cells. A, GHR activation. Serum-starved HEK293-GHR-JAK2 cells were treated without or with GH (500 ng/mL) for 10 minutes, after which detergent cell extracts were immunoprecipitated with anti-GHR or a negative control (nonimmune) antibody (NI). Eluates were separated by SDS-PAGE and immunoblotted with the antiphosphotyrosine (pY) antibody, 4G10. The blot was stripped and reprobed with anti-GHR as a loading control. The fully glycosylated mature form of GHR (m; bracket) and the incompletely glycosylated premature form of GHR (p; arrowhead) are labeled. The immunoblots shown are representative of two such experiments for each condition. B, JAK2 activation. Serum-starved HEK293-GHR-JAK2 cells were treated without or with GH (500 ng/mL) for 10 minutes, after which detergent cell extracts were immunoprecipitated with anti-JAK2. Eluates were separated by SDS-PAGE and immunoblotted with the antiphosphotyrosine (pY) antibody, 4G10. The blot was stripped and reprobed with anti-JAK2 as a loading control. The immunoblots shown are representative of two such experiments for each condition. C, STAT5 activation. Serum-starved HEK293-GHR-JAK2 cells were treated without or with GH (500 ng/mL) for 10 minutes. Detergent cell extracts were resolved by SDS-PAGE and immunoblotted with an antibody that recognizes tyrosine phosphorylated STAT5 (pSTAT5). The blot was stripped and reprobed with antibody for total STAT5 (STAT5) as a loading control. The immunoblots shown are representative of three such experiments for each condition. IP, immunoprecipitation; WB, Western blot.

Differential inducible GHR proteolysis in HEK293-GHR-JAK2 vs C14 cells

In prior work, we demonstrated that treatment with the phorbol ester, PMA, induces rapid and substantial GHR proteolysis in multiple cell lines, including human IM-9 lymphoblasts and human C14 cells (14, 35, 37, 39). To characterize inducible GHR proteolysis in HEK293-GHR-JAK2, we used C14 cells as a positive control in time course (Figure 2, A and B) and PMA concentration dependence (Figure 2, C and D) experiments.

Figure 2.

Figure 2.

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Differential inducible GHR proteolysis in HEK293-GHR-JAK2 vs C14 cells. A, PMA-induced GHR proteolysis (time course). Serum-starved C14 cells (upper panel) or HEK293-GHR-JAK2 cells (lower panel) were treated without or with PMA (0.1 μg/mL) for the indicated durations. Detergent cell extracts of C14 cells were resolved by SDS-PAGE and immunoblotted with anti-GHR. Detergent cell extracts of HEK293-GHR-JAK2 cells were immunoprecipitated with anti-GHRcyt-mAb. Eluates were separated by SDS-PAGE and immunoblotted with anti-GHR. The fully glycosylated mature form of GHR is indicated with a bracket. The immunoblots shown are representative of three such experiments for each condition. B, Densitometric analysis of data from three separate experiments, including that presented in panel A. The GHR level in the absence of PMA treatment is considered 100%. Data are expressed as mean ± SE. C, PMA-induced GHR proteolysis (concentration dependence). Serum-starved C14 cells (upper panel) or HEK293-GHR-JAK2 (lower panel) cells were treated without or with the indicated concentrations of PMA for 15 minutes. Detergent cell extracts of C14 cells were resolved by SDS-PAGE and immunoblotted with anti-GHR. Detergent cell extracts of HEK293-GHR-JAK2 cells were immunoprecipitated with anti-GHRcyt-mAb. Eluates were separated by SDS-PAGE and immunoblotted with anti-GHRcyt-AL47. The fully glycosylated mature form of GHR is indicated with a bracket. The immunoblots shown are representative of three such experiments for each condition. D, Densitometric analysis of data from three separate experiments, including that presented in panel C. The GHR level in the absence of PMA treatment is considered 100%. Data are expressed as mean ± SE. IP, immunoprecipitation; WB, Western blot.

Serum-starved cells were exposed to PMA (0.1 μg/mL) for 5–30 minutes compared with vehicle alone (0 min). For C14 cells, detergent extracts were resolved by SDS-PAGE and blotted with anti-GHR (Figure 2A, upper panel); for HEK293-GHR-JAK2 cells, extracted proteins were first immunoprecipitated with a monoclonal anti-GHR (anti-GHRcyt-mAb) or control immunoglobulin, and eluates were then resolved by SDS-PAGE and blotted with anti-GHR (Figure 2A, lower panel). As anticipated, PMA caused rapid and robust loss of immunodetectable GHR in C14 cells. Densitometric quantitation of multiple independent experiments (Figure 2B) revealed greater than 90% loss of GHR after 30 minutes of PMA exposure. In contrast, PMA induced far less GHR loss (22% at most) in HEK293-GHR-JAK2 cells (Figure 2A, lower panel, and Figure 2B).

For concentration-dependence experiments, cells were treated with PMA for 15 minutes at 0.05, 0.1, 0.2, or 0.5 μg/mL and compared with cells exposed to vehicle (Figure 2C), and GHR abundance was assessed as in Figure 2A. Densitometric evaluation of several such independent experiments (Figure 2D) revealed that even PMA concentrations up to 0.5 μg/mL failed to markedly change GHR abundance in HEK293-GHR-JAK2 cells, whereas GHR loss in C14 cells was easily observed. We note that PMA in concentrations as low as 0.05 μg/mL caused ample phosphorylation of ERK in HEK293-GHR-JAK2 cells (data not shown), indicating that the markedly diminished PMA-induced GHR proteolysis in these cells is not attributable to a defect in the PMA-inducible protein kinase C-ERK activation pathway.

Ratio of steady-state mature TACE to unprocessed TACE precursor (pro-TACE) abundance is greater in C14 cells compared with HEK293-GHR-JAK2 cells

We previously demonstrated that PMA-inducible GHR proteolysis is severely impaired in fibroblasts derived from mice with targeted disruption of the catalytic domain of TACE compared with wild-type TACE-expressing fibroblasts (17). Similarly, a hydroxamate-based chemical inhibitor of metalloproteases, including TACE, blocks inducible GHR proteolysis (14), and reduction of TACE abundance by RNA interference also dramatically lessens PMA-inducible GHR proteolysis (37). These findings suggest that TACE abundance and/or activity are key determinants of susceptibility of GHR to inducible proteolysis. Because the HEK293-GHR-JAK2 cells are dramatically resistant to PMA-inducible GHR proteolysis compared with C14 cells, we compared endogenous TACE abundance in the two cells.

TACE is a transmembrane glycoprotein that can be detected in two forms in cells. The 120-kDa pro-TACE form includes at its extracellular domain N terminus the prodomain, which negatively regulates TACE activity by impacting the nearby catalytic domain (41–43). The prodomain is cleaved after biosynthesis, yielding the 110-kDa mature form of TACE that is the active form enriched on the cell surface (20, 21). To compare the two forms of TACE present in C14 and HEK293-GHR-JAK2 cells, we subjected the immunoprecipitated TACE from each cell to deglycosylation with endoglycosidase H (endoH; removes the high mannose chains characteristic of immature glycoproteins yet to traverse the Golgi apparatus and causes them to migrate more rapidly in SDS-PAGE) vs N-glycosidase F/neuraminidase (F/N; removes carbohydrate chains independent of their content and thus fully deglycosylates independent of maturation stage). As shown in Figure 3A, two forms of TACE protein, labeled pro-TACE and mature TACE, were specifically immunoprecipitated from detergent extracts and detected by immunoblotting for both C14 and HEK293-GHR-JAK2 cells. Consistent with previous reports (44, 45), pro-TACE, but not mature TACE, was sensitive to endoH treatment and both forms were sensitive to F/N. Interestingly, although both forms were present in each cell type, the ratio of mature TACE to pro-TACE differed between them. By densitometric analysis, mature/pro TACE was significantly greater by 2.8-fold on average in C14 cells compared with HEK293-GHR-JAK2 cells. This relatively reduced abundance of the mature (active) form of TACE in HEK293-GHR-JAK2 cells corresponds to the reduced susceptibility of GHR to inducible proteolysis in these cells.

TIMP3 abundance is greater in C14 cells compared with HEK293-GHR-JAK2 cells

TIMP3 is a natural inhibitor of TACE (26). TIMP3's N-terminal domain harbors its inhibitory activity (46), and crystallographic studies demonstrate interaction between TIMP3 and the active site cleft of TACE (47). Although the details of TIMP3's inhibitory mechanisms remain unknown, we considered that, in principle, the differential susceptibility of GHR to inducible, TACE-mediated proteolysis might relate to differences in TIMP3 abundance between the cells. To test this possibility, detergent extracts were subjected to immunoprecipitation with anti-TIMP3 or a control antibody, and eluates were resolved and immunoblotted for TIMP3 (Figure 3C). Notably, TIMP3 was specifically detected in extracts from HEK293-GHR-JAK2 cells, but not from C14 cells, suggesting that endogenous TIMP3 expression is far greater in the cells in which GHR is relatively resistant to inducible proteolysis.

Forced TIMP3 expression in C14 cells reduces the mature TACE to Pro-TACE ratio

Data in Figures 2 and 3 indicate that C14 cells, in which endogenous TIMP3 is low and the mature TACE to pro-TACE ratio is high, exhibit robust inducible GHR proteolysis. In contrast, in HEK293-GHR-JAK2 cells, endogenous TIMP3 is more abundant and the mature TACE to pro-TACE ratio is low and inducible GHR proteolysis is reduced. We further tested this relationship by asking whether the mature TACE to pro-TACE ratio in C14 cells could be altered by forced expression of TIMP3 (Figure 4). Because C14 cells are adenovirally infectable (36), we compared the expression of TIMP3 (Ad-TIMP3) to two negative controls: TIMP2 (Ad-TIMP2) and green fluorescent protein (GFP; Ad-GFP). Detergent extracts were immunoprecipitated with anti-TACE vs nonspecific antibody, and eluted proteins were immunoblotted with anti-TACE. Extracts were also resolved without precipitation and TIMP3 abundance was monitored by immunoblotting (Figure 4A) Expression of TIMP3, but not TIMP2 or GFP, resulted in an approximately 2.3-fold reduced mature TACE to pro-TACE ratio in these cells (Figure 4B). These data strongly suggest that TIMP3 can regulate the availability of active TACE, possibly by regulating TACE maturation at the stage of furin-mediated prodomain removal.

TIMP3 inhibits inducible GHR proteolysis in C14 cells

To test the effects of forced TIMP3 expression on inducible GHR proteolysis in C14 cells, we compared infection of Ad-TIMP3 to Ad-TIMP2 at several (100, 200, and 400) MOIs and, as a further negative control, Ad-GFP at MOI of 400, and the MOI-dependent expression of the TIMPs was verified by specific immunoblotting of cell extracts from Ad-infected C14 cells (Figure 5A). The same cells were serum starved and exposed to PMA (0.1 μg/mL) for 0–60 minutes, and extracts were immunoblotted to detect GHR (Figure 5B). In cells infected with either Ad-GFP or Ad-TIMP2, dramatic PMA-induced loss of GHR (bracket) was observed, indicating neither adenovirus infection itself nor the expression of TIMP2 had any effect on PMA-inducible GHR proteolysis. However, when TIMP3 was expressed, PMA-induced GHR loss was markedly reduced in a MOI-dependent fashion. These data strongly suggest that expression of TIMP3 rendered the cells less sensitive to the PMA-inducible GHR metalloproteolysis.

Figure 5.

Figure 5.

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TIMP3 inhibits inducible GHR proteolysis in C14 cells. A, C14 cells were infected with Ad-GFP, Ad-TIMP2, or Ad-TIMP3 at the indicated MOI. Two days later, serum-starved cells were treated with PMA (0.1 μg/mL) for 15, 30, or 60 minutes. Detergent cell extracts were separated by SDS-PAGE and immunoblotted with anti-TIMP2 (left panel) and anti-TIMP3 (right panel). B, The same detergent extracts from panel A was separated by SDS-PAGE and immunoblotted with anti-GHR. Result from densitometric quantitation of GHR signal is indicated. The GHR signal in the absence of PMA was considered as 1. C, C14 cells were infected with Ad-GFP or Ad-TIMP3 at 400 MOI. Two days later, serum starvation medium from these infected cells were collected and immunoblotted with anti-TIMP3 to verify the presence of TIMP3 in these media. A different pool of serum-starved C14 cells were pretreated for 30 minutes with TIMP3-containing medium or GFP-containing medium as a control, followed by PMA exposure at 0.1 μg/mL for 25 minutes. Detergent cell extract was immunoblotted with anti-GHR for GHR abundance (upper panel) or anti-TIMP3 (lower panel) to evaluate TIMP3 that remained associated with the treated cells. Result from densitometric quantitation of GHR signal is indicated. The GHR signal in the absence of PMA was considered as 1. WB, Western blot.

We sought to also examine the effect of exogenously added, rather than endogenously expressed, TIMP3 on GHR proteolysis in C14 cells. Ad-TIMP3 infection of various cell types is known to yield secreted TIMP3 in the extracellular matrix and/or conditioned medium (48–52). We produced TIMP3-containing conditioned medium by infecting C14 cells with Ad-TIMP3 or, as a control, Ad-GFP and culturing the cells for 2 days in serum-free medium. Immunoblotting of the conditioned medium verified ample TIMP3 (data not shown). A different pool of serum-starved C14 cells were pretreated for 30 minutes with TIMP3-containing medium vs GFP-containing medium and then exposed to PMA (0.1 μg/mL) for 25 minutes. Detergent cell extracts were immunoblotted with anti-GHR (Figure 5C, upper panel). When the cells were pretreated with GFP-containing medium, PMA induced a marked loss of GHR. In contrast, there was minimal change in GHR abundance in cells that were treated with TIMP3-containing medium. Interestingly, TIMP3 was detected by immunoblotting (Figure 5C, lower panel) of detergent extracts of the cells that were treated with TIMP3-containing conditioned medium but not GFP-containing medium. Thus, TIMP3, originating from the conditioned medium, remained associated with the treated cells when they were harvested. These results suggest TIMP3, when applied exogenously, can inhibit PMA-inducible GHR proteolysis, possibly through inhibition of TACE activity.

TIMP3 expression prevents PMA-induced desensitization to GH in C14 cells

Our previous cell culture and in vivo findings indicate that inducible GHR proteolysis modulates GH sensitivity (17–19). Because our current findings strongly suggest TIMP3 as a regulator of GHR metalloproteolysis and thus cell surface GHR abundance, we next asked whether TIMP3 expression in C14 cells can preserve GH sensitivity in the face of PMA exposure (Figure 6).

Figure 6.

Figure 6.

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TIMP3 expression prevents PMA-induced desensitization to GH in C14 cells. A, C14 cells were infected with Ad-TIMP3 or Ad-GFP as a control (both at 400 MOI). Two days later, serum-starved cells were treated with PMA (0.1 μg/mL) for 30 minutes, followed by GH treatment at 500 ng/mL for 10 minutes. Detergent cell extracts were separated by SDS-PAGE and immunoblotted with anti-GHR for GHR abundance or anti-TIMP3 to confirm TIMP3 expression. Detergent cell extracts were also sequentially immunoblotted with anti-pSTAT5 and anti-STAT5. Result from densitometric quantitation of GHR signal is indicated. The GHR signal in the absence of PMA and GH (lane 1) was considered as 1. Densitometric quantitation of pSTAT5/STAT5 (p/t) is also shown, and GH-induced pSTAT5/STAT5 signal in the absence of PMA was considered as 1 (lane 2). B, The same treatment was carried out as in panel A. Detergent cell extracts were separated by SDS-PAGE and immunoblotted with anti-GHR for GHR abundance or anti-TIMP3 to confirm TIMP3 expression. Detergent cell extracts were immunoprecipitated with anti-JAK2. Eluates were separated by SDS-PAGE and immunoblotted with antiphosphotyrosine (pY) antibody, 4G10, then stripped and reprobed with anti-JAK2. IP, immunoprecipitation; WB, Western blot.

We first examined GH-induced STAT5 phosphorylation (Figure 6A). C14 cells were infected with Ad-TIMP3 or Ad-GFP as a control (both at 400 MOI). Two days later, cells were treated sequentially with PMA vs vehicle for 30 minutes and then for 10 minutes with GH vs vehicle, and detergent cell extracts were examined by immunoblotting. This confirmed that PMA caused substantial GHR loss in cells infected with Ad-GFP but that TIMP3 expression largely prevented this effect of PMA, as expected. Corresponding to the PMA-induced lessening of GHR, GH-induced STAT5 phosphorylation was also dramatically reduced in Ad-GFP-infected cells pretreated with PMA. In contrast, GH-induced acute STAT5 phosphorylation was not diminished in the PMA-pretreated samples when cells were infected with Ad-TIMP3. GH-induced JAK2 activation was examined in a separate similar experiment, in which JAK2 was immunoprecipitated and anti-pTyr blotting identified the tyrosine phosphorylated kinase (Figure 6B). Again, GH-induced JAK2 phosphorylation was markedly reduced in Ad-GFP-infected cells pretreated with PMA, concordant with PMA-induced loss of GHR in those cells. However, TIMP3 expression rendered the cells resistant to both PMA-induced GHR loss and PMA-induced desensitization to GH-induced JAK2 phosphorylation. These data bolster the notion that GHR abundance is a key determinant of GH sensitivity and implicate TIMP3 as a novel modulator of JAK2/STAT5 pathway responsiveness to GH.

Silencing of TIMP3 in HEK239-GHR-JAK2 cells yields reduced GHR abundance and diminished GH sensitivity

We pursued further the idea that TIMP3 can regulate GH sensitivity by assessing the effect of reducing TIMP3 expression in HEK293-GHR-JAK2 cells, in which TIMP3 is relatively abundant (Figure 7). Cells were transfected with a well-validated (33) TIMP3 siRNA vs a control siRNA. Immunoprecipitation and immunoblotting of cell extracts verified substantial knockdown of TIMP3 protein by the specific siRNA compared with the control (Figure 7A, top panel). Notably, our densitometric evaluation of the GHR levels by immunoprecipitation and immunoblotting with anti-GHR revealed that mature GHR abundance was reduced by 32% in the TIMP3 siRNA-transfected cells vs the cells transfected with control siRNA (Figure 7A, middle and bottom panels; for densitometry, n = 4 experiments and P < .02). Thus, reduction of TIMP3 in these cells that express relatively lower GHR than do C14 cells lessened mature GHR abundance, suggesting TIMP3 may modulate cell surface GHR availability.

Figure 7.

Figure 7.

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Silencing of TIMP3 in HEK239-GHR-JAK2 cells yields reduced GHR abundance and diminished GH sensitivity. A, HEK293-GHR-JAK2 cells were transfected with control or TIMP3 siRNA as described in Materials and Methods. Three days later, detergent cell extracts were immunoprecipitated and immunoblotted with anti-TIMP3 to confirm TIMP3 knockdown. Detergent cell extracts were immunoprecipitated with anti-GHRcyt-mAb and immunoblotted with anti-GHR to evaluate GHR abundance. The fully glycosylated mature form of GHR (m; bracket) and the incompletely glycosylated premature form of GHR (p; arrowhead) are labeled. The immunoblots shown are representative of four such experiments. Densitometric analysis of data from four separate experiments is shown (lower panel). Data are expressed as mean ± SE. B, GH-induced STAT5 phosphorylation in HEK293-GHR-JAK2 cells. Serum-starved HEK293-GHR-JAK2 cells were treated without or with the indicated concentrations of GH for 10 minutes. Detergent cell extracts were resolved by SDS-PAGE and sequentially immunoblotted with anti-pSTAT5 and anti-STAT5. C, Three days after siRNA transfection, serum-starved HEK293-GHR-JAK2 cells were treated with GH (50 ng/mL) for 10 minutes. Detergent cell extracts were immunoblotted with anti-pSTAT5 and then stripped and reprobed with anti-STAT5 as a loading control. Densitometric analysis of data from three separate experiments, including that presented in panel C, is shown (lower panel). Data are expressed as mean ± SE. IP, immunoprecipitation; WB, Western blot.

We reasoned that the lessened mature GHR levels in TIMP3 siRNA-transfected cells would affect GH sensitivity. To approach this issue, we first evaluated the GH concentration dependence for STAT5 phosphorylation in HEK293-GHR-JAK2 cells (Figure 7B). Treatment with GH at 10 ng/mL for 10 minutes allowed detectable STAT5 phosphorylation and maximal stimulation was reached with 100–250 ng/mL GH. To test the effect of TIMP3 silencing, we thus chose 50 ng/mL GH as a submaximal concentration (Figure 7C, upper panel). Notably, under these conditions, GH-induced STAT5 phosphorylation was reduced by 37% in TIMP3 siRNA-transfected cells compared with control siRNA-transfected cells when analyzed densitometrically (n = 3; P < .01). Taken together, the data in Figure 7 indicate that silencing of TIMP3 in HEK293-GHR-JAK2 cells reduced mature surface GHR abundance (likely by enhanced constitutive GHR metalloproteolysis) and thereby diminished GH sensitivity in terms of acute STAT5 phosphorylation.

Discussion

GHR from various species is subject to TACE-mediated metalloproteolysis in the receptor's membrane-proximal extracellular domain (14–18, 53). This cleavage could potentially modulate GH action in several ways. Shedding of the extracellular domain contributes to the circulating pool of high affinity GHBP, which itself can modulate GH stability, clearance, and local availability (11, 54–58). GHR metalloproteolysis may also contribute to the regulation of steady-state GHR abundance, complementing other constitutive regulatory parameters such as the rates of GHR gene transcription, GHR biosynthesis, and basal GHR endocytosis and degradation to determine the GHR's half-life and cell surface availability (37, 59). Additionally, rapid increases or decreases of GHR metalloproteolysis could reflect physiologic or pathophysiological processes, allowing dynamic regulation of GHR availability and hence cellular GH sensitivity (11, 19). In each of these situations, TACE availability and activity are thus important determinants that allow or promote modulation of GH action.

Because TACE has a range of physiologically relevant substrates, mechanisms of TACE maturation, activation, and regulation have been extensively studied. TACE itself undergoes regulated cleavage to yield its mature (active) form. Removal of the extracellular prodomain is critical in this process and is catalyzed by the proprotein convertase, furin, via a recognition sequence (RXXR) that resides between the prodomain and the catalytic domain (21, 60, 61). Mature TACE is EndoH resistant, consistent with prodomain removal taking place in the late Golgi, the primary subcellular locale of furin (44, 62, 63). Recent studies also point to the importance of the inactive Rhomboid family member, iRhom2, in allowing proper TACE maturation and surface presentation (45, 64, 65) and perhaps in influencing the specificity of certain TACE substrates (66, 67). TACE is activated upon stimulation of the ERK or p38 MAPK pathway by extracellular signals, including growth factors, inflammatory mediators, and stress (68, 69). Activation of the ERK and/or p38 MAPK pathways results in the phosphorylation of TACE (70–73), but how these changes lead to TACE activation is not clear. TIMPs regulate the activity of metalloproteases by forming tight, noncovalent complexes in a 1:1 stoichiometry (25). TIMP3 is unique among TIMPs in that it binds to the extracellular matrix (74–76) and is a natural inhibitor of TACE (26, 27). TIMP3 interacts with the TACE extracellular domain (47). Recent studies suggest TACE and TIMP3 are associated at the cell surface and that TACE activation may decrease this interaction (77).

In this study, we pursued the functional relationships between TACE, TIMP3, GHR abundance, and cellular GH sensitivity. We compared two model cell culture lines, C14 and HEK293-GHR-JAK2, in terms of their propensity for inducible GHR proteolysis and found that PMA induced far greater GHR loss in C14 than in HEK293-GHR-JAK2 cells. This greater sensitivity to inducible proteolysis corresponded to an enhanced mature/pro ratio of TACE along with markedly reduced TIMP3 abundance in C14 cells. Forced expression of TIMP3 specifically reversed the mature to pro-TACE ratio in C14 cells, at the same time rendering GHR less susceptible to PMA-induced proteolysis and preventing the desensitization to GH caused by PMA treatment in the TIMP3-deficient C14 cells. Notably, the inhibitory effect of TIMP3 on susceptibility of GHR to proteolysis was conferred both by TIMP3 expression in C14 cells and by the addition of TIMP3-containing conditioned medium to the cells. In contrast to the findings in C14 cells, siRNA-mediated silencing of TIMP3 in HEK293-GHR-JAK2 cells reduced the abundance of the mature GHR and desensitized these cells to GH stimulation in terms of acute STAT5 phosphorylation.

Our findings extend our understanding of the regulation of cellular GH sensitivity in several ways. The finding that the endogenous TACE regulator, TIMP3, can modulate acute GH-induced signaling validates our previous findings with a chemical TACE inhibitor (18) and further strengthens the hypothesis that regulation of constitutive and inducible GHR proteolysis, and thereby cell surface GHR abundance, is a meaningful posttranscriptional mechanism regulating GH action. Thus, physiological alterations and pathophysiological states, such as inflammation, could rapidly modulate GH sensitivity in various cells and tissues by this mechanism more nimbly than by changes in gene expression of either the GHR or its signal transducing or regulatory elements. Furthermore, the effects of TIMP3 in conditioned medium suggest that local and/or humoral communication between cells and tissues via this matrix-associated inhibitor could broaden the universe of influences on GH-sensitive target cells in important ways.

The findings that TIMP3, either expressed in C14 cells or added to C14 cells as conditioned medium, suppresses GHR metalloproteolysis are consistent with accepted mechanisms of TIMP3 inhibition of TACE action at the cell surface. In addition, however, we also found that expression of TIMP3 in C14 cells significantly reduced the mature to pro ratio of TACE. Notably, we did not see an effect of adding TIMP3-enriched conditioned medium on the C14 cell mature to pro ratio (data not shown), suggesting that the effect of TIMP3 on modulating TACE maturation may occur within the cell, perhaps during TACE biosynthesis. The proposed two distinct mechanisms for regulation of TACE by TIMP3 are illustrated in Figure 8. Using primary isolated placental trophoblasts, Ma et al (78) reported that a reduction in TIMP3 resulted in increased detection of TACE. However, the TACE form (mature vs pro) that was affected was not determined, precluding direct comparison with our findings of TIMP3-dependent alteration of the mature to pro TACE ratio. It has also been reported that general inhibition of proprotein convertase activity reduces the mature to pro TACE ratio (60). Thus, our findings raise the intriguing possibility that TIMP3, at least when overexpressed, may affect intracellular TACE processing, perhaps by influencing proprotein convertase activity. This idea warrants further exploration. TIMP3 is known to interact with and inhibit proteins other than TACE, such as ADAM10, ADAM12, and ADAM-TS4 (79). We have previously reported ADAM10 also contributes to inducible GHR proteolysis (37). Therefore, it is possible that TIMP3 could also, at least partially, control GHR abundance through the interaction with one of these other proteases.

Figure 8.

Figure 8.

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Mode of inhibition of TACE-mediated inducible GHR proteolysis by TIMP3. A, At a low level of TIMP3, prodomain of TACE is removed, mediated by furin proteases, and mature TACE reaches the cell surface. In response to stimuli such as PMA, TACE is activated and mediates GHR proteolysis. Cell surface GHR abundance decreases, and GH sensitivity is reduced. B, Higher level of TIMP3 blocks TACE maturation by inhibition of the prodomain removal, which results in reduced mature to pro ratio of TACE. In the extracellular matrix, secreted TIMP3 binds and keeps cell surface mature TACE inactive. TACE is therefore suppressed via two distinct mechanisms, resulting in maintained cell surface GHR abundance and preserved GH sensitivity.

Key requirements for GH-triggered signaling, GHR, JAK2, STAT5, other pathways, and other kinases and phosphatases, have been elucidated over the past 3 decades. To explore and exploit the physiology of GH action, future studies will increasingly address the mechanisms that more subtly modulate cellular and tissue sensitivity to GH under varied conditions. The data presented herein enrich our understanding of proteolytic mechanisms that can rapidly regulate GHR abundance via the TACE/TIMP3 nexus and provide a framework for future studies that address this regulatory mechanism in vivo.

Acknowledgments

We acknowledge the generous provision of reagents by those named in the text. We also appreciate the helpful conversations with Dr R. A. Black and members of the Frank laboratory.

Parts of this work were presented at the 94th Annual Meeting of The Endocrine Society, Houston, TX, 2012.

This work was supported by a Veterans Affairs Merit Review Award (to S.J.F.) and National Institutes of Health Grants DK46395 and DK58259 (to S.J.F.).

Disclosure Summary: The authors have nothing to disclose.

Footnotes

Abbreviations:
Ad
adenovirus
ADAM
a disintegrin and metalloprotease
endoH
endoglycosidase H
F/N
N-glycosidase F/neuraminidase
GFP
green fluorescent protein
GHBP
GH binding protein
GHR
GH receptor
HEK293
human embryonic kidney 293
JAK
Janus kinase
MOI
multiplicity of infection
PMA
phorbol-12-myristate-12-acetate
pro-TACE
unprocessed TACE precursor
SDS
sodium dodecyl sulfate
(msiRNA
small interfering RNA
STAT
signal transducer and activator of transcription
TACE
TNF-α converting enzyme
TIMP
tissue inhibitors of metalloprotease.

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