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5 Results
5.1 SUBCELLULAR LOCALIZATION OF HYALURONAN SYNTHASES (I)
5.1.1 Immunostainings of tissues
Because hyaluronan content of the dermis is high, human skin sections were used as an example to test the localization and amount of HAS isoenzymes in human tissues (I, Figure 5). Dermal fibroblasts were intensely stained with all HAS antibodies. Most of the staining detected was cytoplasmic, but also plasma membrane signal could be detected, for example for HAS1 (I, Figure 5b).
5.1.2 Immunostainings of cultured cells
We used three different cell lines, human skin dermal fibroblasts, human keratinocytes (HaCat) and transformed fibroblastic like COS-‐‑1 cells derived from monkey kidney, to study subcellular localization of endogenous HASs. These cell lines produce different amounts of hyaluronan, the fibroblasts producing the highest and COS-‐‑1 cells the lowest amount (I, table 1). In line with their high hyaluronan production (Jokela et al. 2013) fibroblasts were intensely stained for all HAS isoenzymes with the antibodies used (I, Figure 7a-‐‑c). The signal for HAS1 was low in HaCat keratinocytes, while HAS2 and HAS3 immunostainings were clearly positive, in accordance with the substantial levels of HAS2 and HAS3, and low HAS1 mRNA in these cells (Saavalainen et al. 2007) (I, Figure 7d-‐‑f). The immunostainings for all HASs were almost negative in COS-‐‑1 cells (I, Figure 7g-‐‑i).
HAS3 was abundant in plasma membrane and the protrusions of the cells. These areas were also rich in hyaluronan (I, Figure 8j-‐‑l). The majority of HAS1 was found intracellularly, mostly in the Golgi area. A weak signal was also seen in the plasma membrane and its protrusions (I, Figure 8a-‐‑c). Cytoplasmic vesicles contained most of the HAS2 staining (I, Figure 8d-‐‑f). Interestingly, a part of the HAS2 signal was also localized in the ER and nuclear membrane, especially in the fibroblasts (I, Figure 7b).
Native MCF-‐‑7 cells, expressing a low level of HAS3 mRNA, a modest level of HAS2, and almost no HAS1, produce about 2.6 ng hyaluronan/10,000cells/24h (Kultti et al. 2009), while the MCF-‐‑7 cells transfected with HAS1-‐‑3 constructs synthesize large quantities of hyaluronan (Kultti et al. 2009). The antibody for HAS1 stained the HAS1 overexpressing cells nicely, but produced no signal in cells overexpressing HAS2 and HAS3 (I, Figure 6).
Likewise, the antibody for HAS2 showed no cross-‐‑reaction in cells overexpressing HAS1 and HAS3. No cross-‐‑reactivity was seen with HAS3 antibody either.
5.1.3 GFP-‐‑tagged HAS proteins
To confirm the different subcellular distributions of the HAS isoforms, as suggested by the immunostainings, MCF-‐‑7 cells were transiently tranfected with the human HAS-‐‑GFP-‐‑
constructs. Each of the GFP-‐‑tagged isoenzymes had a typical subcellular distribution (I, Figure 9). GFP-‐‑HAS1 showed the lowest signal in the plasma membrane. It was especially abundant in the Golgi area and in the ER. However, some GFP-‐‑HAS1 positive intracellular vesicles were seen near the plasma membrane and in the thin protrusions of the plasma membrane (I, Figure 9a,b). The GFP-‐‑HAS2 signal was primarily found in the ER, Golgi and
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cytoplasmic vesicles. Some of the protrusions of the plasma membrane, especially those standing on the edges of lamellipodia, contained high levels of GFP-‐‑HAS2 (I, Figure 9c,d).
GFP-‐‑HAS3 had the highest plasma membrane signal among the HASs, in addition to the intracellular locations similar to the other HAS isoenzymes. The apical surface of the GFP-‐‑
HAS3 expressing cells was covered by very long plasma membrane protrusions positive for GFP-‐‑HAS3 (I, figure 9e,f).
5.2 SPATIAL AND TEMPORAL DISTRIBUTION OF HYALURONAN AND HAS1-3 DURING MOUSE EMBRYONIC DEVELOPMENT (I)
Paraffin sections from mouse embryos at different ages were stained with bHABC and HAS antibodies to detect hyaluronan and HAS1-‐‑3. The specificity of the hyaluronan staining was controlled by treatment of the sections with hyaluronidase, and blocking the probe with hyaluronan oligosaccharides. The spesificities of the HAS1-‐‑3 stainings were determined by blocking with peptides corresponding to those used for immunization (I, Figure2, i-‐‑l) or replacing the primary antibody with non-‐‑immune IgG.
The stainings for hyaluronan were typically intense in all stages of the development (I, Figures 1-‐‑4). Hyaluronan was abundant especially in the tissues of mesodermal origin, like the stuctures surrounding the neural tube, branchial arch, cardic tube and its cushions in samples from the E9 embryos (I, Figure 1a-‐‑d). During the embryonic day11 (E11)-‐‑E15 stage HA accumulated in the mesenchymal tissues all over the body (I, Figures 2m, 3a), in the skin and its underlying connective tissues (I, Figure 2e, Figure 3a,c), cartilage (I, Figure 3m) and certain brain areas (I, Figure 3a,q). In the E17 stage hyaluronan stainings were still intense in the connective tissues, part of the brain, kidney and developing eye (I, Figure 4a,e,I,m). Interestingly, at this stage parts of the brain, liver and calcified bones were almost negative for hyaluronan (Figure 4).
As expected, the HAS stainings in the embryos were localized in the cells (as opposed to the extracellular matrix). The main location was cytoplasm for all HASs (I, Figures 1-‐‑4).
Nevertheless, HAS2 signal was seen in positions consistent with plasma membrane in the tubular epithelium of the developing kidneys (I, Figure 4o). In addition, some plasma membrane protrusions on the mesenchymal cells of the intramembraneous bones were stained with the HAS1 and HAS3 antibodies (I, Figures 2n,p, arrows). In general, the mesenchymal cells in particular were highly positive for all HASs (I, Figures 2n-‐‑p), but the staining was also high in the developing epidermal keratinocytes. In the kidney, the stainings of the HASs were most intense in the tubular cells (I, Figures 4n-‐‑p). The sections through a whole E17 mouse embryo stained for HAS1, 2 and 3 and hyaluronan, show a relatively high signal intensity of all HASs e.g. in the developing skin and cartilage, as well as the overall distribution of each isoenzyme (Figure 4). Large amounts of hyaluronan are found especially in the connective tissues, brain and kidney (I: Figure 4).
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Figure 4. Distribution of hyaluronan and hyaluronan synthases in sections of a whole mouse E17 embryo. The sections were stained with bHABC to detect hyaluronan and with affinity purified polyclonal antibodies for HAS1, HAS2 and HAS3. The brown color indicates the signal for hyaluronan and the antibodies, and hematoxylin was utilized to stain the nuclei (blue). Magnification bar 1 mm.
The staining intensity of HAS2 was generally highest in the E9 embryos, but signals were present also for HAS1 and HAS3 (I, Figure 1). On the embryonic day 11 all HASs stained with high intensity in the heart, and mesenchymal tissues such as dermis. At this stage, a stronger staining intensity was found for HAS1 and HAS3 than HAS2 in the developing bones. Epithelial tissues were mostly negative for all HASs (I; Figure 2). In E15 chondrocytes the interior of the cartilage was intensively stained for HAS1-‐‑3. The stratified epithelia of mouth and skin had turned positive for HASs (I, Figure 3). In stage E17 all HASs were positive in the mesenchymal tissues such as skin. At this stage, there was less HAS1 than HAS2 and HAS3 in the epidermis (I, Figure 4).
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5.3 HYALURONAN, CD44 AND HAS1-3 IN UV RADIATED MOUSE EPIDERMIS (II)
5.3.1 Hyaluronan and CD44 stainings
In paper (II) mice were exposed to UV lamps simulating the spectrum of the sun (280-‐‑400 nm) for 35min three times a week for 10.5 months, with doses corresponding to one human minimum erythema dose (MED). Most of the treated animals showed epidermal thickening as compared with non-‐‑exposed controls. There was a squamous cell carcinoma in every fifth, and dysplastic changes in every third treated sample. No dysplasias or squamous cell carcinomas were found in control animals (II, Table 1).
Normal adult mouse epidermis was mostly negative for hyaluronan, but the dermal connective tissue showed a highly intensive staining (II, Figure 1a). There was a faint signal for CD44 in the interfollicular epidermis, especially in the basal layers (II, Figure 1b). The treatment with UV caused hyaluronan accumulation in the epidermis, especially in the areas with strong hyperplasia (II, Figure 1e). The epidermal area positive for hyaluronan, and the intensity of the staining increased in hyperplasia. There was a positive correlation between the degree of hyperplasia and level of hyaluronan. The number of cell layers positive for hyaluronan also increased with advancing hyperplasia (II, Figure 1c,e). The benign hyperplasia in mouse epidermis displayed a hyaluronan distribution similar to that of the human epidermis, both containing hyaluronan between the basal and spinous layers, but missing hyaluronan in the granular and more superficial layers (II, Figure 1c). The staining of CD44 also increased with hyperplasia (II, Figure 1f). The dysplastic and squamous carcinoma cells were moderately or intensely positive for hyaluronan and CD44, with occasional areas of a faint signal (II, Figure 2).
Dermis was positive for hyaluronan in both control and UV-‐‑treated samples.
Hyaluronan staining under the epidermal basement membrane was reduced in 69% of the hyperplastic samples (II, Figure 1).
5.3.2 HAS immunostainings
Normal mouse epidermis was almost negative for all HASs with only a few faintly positive cells. Dermal fibroblasts had a weak or moderate staining for all HASs (II, Figures 3a,e,i).
The UV-‐‑treatment increased the HAS1, HAS2 and HAS3 signals both in the epidermis and dermis (II, Figure 3). The response was strongest for the HAS2 immunostaining.
5.4 HYALURONAN, CD44 AND HAS1-3 IN LUNG MESOTHELIOMA AND ADENOCARCINOMA (III)
5.4.1 Hyaluronan staining in mesotheliomas and adenocarcinomas
Hyaluronan was observed on the surfaces of the mesothelioma cells and in the adjacent extracellular space (III, Figures 1 a, b, d, e). There was also some intracellular staining. The intensity of the staining and its coverage are varied between the samples. The staining intensity was weak or moderate in most of the mesothelioma cases. Adenocarcinoma cells had significantly less (p=0.001) hyaluronan than mesothelioma cells (III, Figures c, f). The stromal tissue was moderately positive for hyaluronan in both mesotheliomas and adenocarcinomas.
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Most cancer cells and stromal cells were CD44 positive in the epithelioid mesotheliomas (III, Figure 3 a, d). In some of the sarcomatoid mesotheliomas most of the cancer cells were positive for CD44 while in some cases only a small part of the cells were positive (III, Figures 3 d, e). The staining of CD44 in the stromal cells was generally less intense. A part of the adenocarcinoma cells was almost negative for CD44 while a part of these cells showed a faint signal (III, Figures e, f). There were no significant differences in the cancer cell or the stromal CD44 staining intensity between mesotheliomas and adenocarcinomas (III, Figure 3).
5.4.3 HAS immunostainings
The stromal cells in both mesothelioma and adenocarcinoma samples were mostly negative, or weakly positive for all HAS isoenzymes (III, Figure 5). There were HAS positive cancer cells in epithelioid mesotheliomas and adenocarcinomas (III, Figure 5a, c), but in the sarcomatoid mesotheliomas the cancer cells were weakly stained (III, Figures 5b, e, h). The granular staining was found intracellularly, while only a few cells showed a HAS positive plasma membrane (III, Figures 5c, f, I). The differences in the intensity or frequency of the HAS stainings between mesotheliomas and adenocarcinomas were not statistically significant.
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