High molecular weight isoforms of growth hormone in cells of the immune system

Abstract

A substantial body of research exists to support the idea that cells of the immune system produce growth hormone (GH). However, the structure and mechanism of action of lymphocyte-derived GH continues to remain largely unknown. Here we present the results of Western analysis of whole cell extracts showing that different molecular weight isoforms of GH of approximately 100, 65, and 48 kDa can be detected in primary mouse cells of the immune system and in the mouse EL4 cell line. The identity of the 65 and 48 kDa isoforms of GH were confirmed by mass spectrometry. The various isoforms were detected in both enriched T and B spleen cell populations. The large molecular weight isoform appears to reside primarily in the cytoplasm, whereas the lower molecular weight 65 and 48 kDa isoforms were detected primarily in the nucleus. These results also suggest that GH isoforms are induced by oxidative stress. In EL4 cells overexpressing GH, the expression of luciferase controlled by a promoter containing the antioxidant response element is increased almost threefold above control. The data suggest that the induction of isoforms of the GH molecule in cells of the immune system may be an important mechanism of adaptation and/or protection of lymphoid cells under conditions of oxidative stress.

Fig. 1

GH protein expression in EL4 cells and primary mouse spleen cells. Whole cell extracts were prepared from EL4 cells (lane 1) and primary mouse spleen cells (lane 2) as described in the methods. After SDS-PAGE (8%) and transfer to PVDF membranes, Western blot analysis was performed using commercial Ab to GH (Santa Cruz) and bands visualized using a chemiluminescence substrate for HRP (GE Healthcare). The approximate molecular weight for isoforms of GH are shown with arrows on the right. The results shown above are typical of an experiment repeated 5 times (*p<0.05).

Fig. 2

GH protein expression in primary mouse spleen, mouse T, and mouse B cells. Whole cell extracts were prepared from unfractionated spleen cells and nylon column purified T and B cells as described in the methods. After SDS-PAGE (8%) and transfer to PVDF membranes, Western blot analysis was performed using commercial Ab to GH (Santa Cruz) and bands visualized using a chemiluminenscence substrate for HRP (GE Healthcare). The approximate molecular weight for isoforms of GH are shown with arrows on the right. Key: Lane 1 (unfractionated spleen cells); lane 2 (T cells); lane 3 (B cells). The results shown above are typical of an experiment repeated 5 times (*p<0.05).

Fig. 3

NanoLC-tandem mass spectra of lymphocyte mouse GH.

Fig. 4

Lymphocyte GH in enriched cytoplasmic and nuclear fractions of EL4 cells. Subcellular fractions were prepared as described in the methods. After SDS-PAGE (8%) and transfer to PVDF membrane, Western blot analysis was performed using commercial Ab to GH (Santa Cruz) and bands visualized using a chemiluminescence substrate for HRP. Blots were stripped and reprobed with specific Abs to actin or proliferating cell nuclear antigen (PCNA). Key: Lane 1 (cytoplasm); lane 2 (cytoplasmic membrane), lane 3 (nucleus); lane 4 (nuclear membrane). Approximate molecular weight for isoforms of GH are shown with arrows on the left. The results shown above are typical of an experiment repeated 5 times.

Fig. 5

Lymphocyte GH in enriched cytoplasmic and nuclear fractions of mouse spleen cells. Subcellular fractions were prepared as described in the methods. After SDS-PAGE (8%) and transfer to PVDF membrane, Western blot analysis was performed using commercial Ab to GH (Santa Cruz) and bands visualized using a chemiluminescence substrate for HRP. Blots shown below were stripped and reprobed with specific Abs to actin (lanes 1,2,4,5) or PCNA (lanes 3,6). The approximate molecular weight for isoforms of GH are shown with arrows on the right. Key; Lane 1 (T cells, whole cell); lane 2 (T cells, cytoplasm), lane 3 (T cells, nucleus); lane 4 (B cells, whole cell); lane 5 (B cells, cytoplasm); lane 6 (B cells, nucleus). Asterisks (*) denote a significant difference (p<0.05) in the T cell nuclear fraction to the whole T cell fraction and between the B cell nuclear fraction to the T cell nuclear fraction. The results shown above are typical of an experiment repeated 5 times.

Fig. 6

GH protein expression in mouse spleen cells treated with PDTC. Cells were treated for 16 hrs with different doses of PDTC after which whole cell extracts were prepared as described in the methods. After SDS-PAGE (8%) and transfer to PVDF membranes, Western blot analysis was performed using commercial Ab to GH (Santa Cruz) and bands visualized using a chemiluminence substrate for HRP. Blots were stripped and reprobed with specific Abs to actin. Asterisks (*) denote a significant difference (p<0.05) from control GH isoforms. The results shown are typical of an experiment repeated 3 times. Key: Lane 1 (nontreated control); lane 2 (PDTC, 25 µM); lane 3 (PDTC, 50 µM); lane 4 (PDTC, 100 µM).

Fig. 7

GH protein expression in mouse spleen cells treated with DEM. Cells were treated for 16 hrs with different doses of DEM after which whole cell extracts were prepared as described in the methods. After SDS-PAGE (8%) and transfer to PVDF membranes, Western blot analysis was performed using commercial Ab to GH (Santa Cruz) and bands visualized using a chemiluminence substrate for HRP. Blots were stripped and reprobed with specific Abs to actin. Asterisks (*) denote a significant difference (p<0.05) from control. The approximate molecular weights for isoforms of GH are shown with arrows on the right. The results shown are typical of an experiment repeated 3 times. Key: Lane 1 (nontreated control); lane 2 (DEM, 1400 µM); lane 3 (DEM, 700 µM); lane 4 (DEM, 350 µM); lane 5 (DEM, 175 µM); lane 6 (DEM, 87.5 µM).

Fig. 8

Antioxidant promoter activity in vector alone control and GHo cells. EL4 cells were transfected by electroporation as described in the methods section with control Ti/Lµc, ARE Luc, and β-Gal reporter constructs. Twenty-four hours later, cells were harvested and the expression of luciferase measured in cell extracts and normalized by β-Gal. The results represent means ± SEM from three separate experiments (* p<0.05).